Corn transgenic event MON 95379 and methods for detection and uses thereof

ABSTRACT

The invention provides a transgenic corn event MON 95379, plants, plant cells, seeds, plant parts, progeny plants, and commodity products comprising event MON 95379. The invention also provides polynucleotides specific for event MON 95379 and methods for using and detecting event MON 95379 as well as plants, plant cells, seeds, plant pails, progeny plants, and commodity products comprising event MON 95379.

REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.16/525,278, filed Jul. 29, 2019 (pending), which claims the benefit ofU.S. provisional application No. 62/711,810, filed Jul. 30, 2018, eachof the disclosures of which is herein incorporated by reference in itsentirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file named MONS463US_ST25 is 74.7kilobytes (measured in Microsoft Windows®), was created on Jul. 25,2019, is filed herewith by electronic submission, and is incorporated byreference.

FIELD OF THE INVENTION

The invention relates to recombinant DNA molecules present in and/orisolated from corn event MON 95379. The invention also relates totransgenic corn plants, plant parts, and seeds, cells, and agriculturalproducts containing corn event MON 95379, as well as methods of usingthe same and detecting the presence of corn event MON 95379. Transgeniccorn plants, parts, seeds and cells containing corn event MON 95379 DNAexhibit resistance to insect infestations in the family Lepidoptera.

BACKGROUND OF THE INVENTION

Corn (Zea mays) is an important crop and is a primary food source inmany areas of the world. The methods of biotechnology have been appliedto corn for improvement of the agronomic traits and quality of theproduct. One such agronomic trait is insect resistance, which isaccomplished through the expression of heterologous insect toxins, alsoknown as transgenes, inserted into the genome of the corn plant.

The expression of such transgenes in a transgenic plant, plant part,seed or cell may be influenced by many different factors, including theelements used in the cassettes driving transgene expression and theinteraction of those elements within a cassette. This is complicatedfurther for a transgenic insertion containing two or more expressioncassettes, with each expression cassette having a transgene conferring aseparate trait, also known as a multi-gene transgenic event. Acommercially useful multi-gene transgenic event requires that each ofthe transgenes in the transgenic insertion express in the mannernecessary for each trait. To achieve this, individual expressioncassettes first are designed and tested in plants, and the expressioncassettes that show the best insect activity, while not resulting innegative phenotypes due to expression, are selected for each trait.Next, the selected expression cassettes for one trait are combined withthe selected expression cassettes for the other trait into a singleconstruct. Multiple constructs are designed using different orientationsto provide the best resistance and prevent the occurrence of negativephenotypes or negative agronomics, such as lower yield. The constructsare tested to ensure all the expression cassettes function well togetherand each transgene is properly expressed. Then, the selected combinationand orientation of expression cassettes is used as a single transgenicinsert to produce hundreds transgenic events, each event being theresult of the random insertion of the construct in a different genomiclocation.

Each transgenic event is unique in its molecular profile and chromosomalinsertion point. Because of the variability involved in event creation,each unique event must be analyzed through multiple generations ofplants—in each step assessing the molecular characterization, proteinexpression efficacy, and agronomics—to select a superior event forcommercial use. The performance of an event in a transgenic plant, plantpart, seed or cell, and therefore its effectiveness, may be influencedby the genomic location of the transgene insertion. Specifically, theeffectiveness of the event can be impacted by cis and/or trans factorsrelative to the integration site or chromatin structure. Events can havethe same transgenic insertion and nonetheless have different transgeneexpression levels and performance across tissues and developmentalstages, in various germplasm, or under specific growth conditions. Theremay also be undesirable phenotypic or agronomic differences between someevents. Therefore, it is necessary to produce and analyze a large numberof individual plant transformation events in order to select an eventhaving superior properties relative to the desirable trait, and theoptimal phenotypic and agricultural characteristics necessary to make itsuitable for commercial purposes. Further, the creation of a multi-geneevent for commercial use requires rigorous molecular characterization,greenhouse testing, and field trials over multiple years, in multiplelocations, and under a variety of conditions so extensive agronomic,phenotypic, and molecular data may be collected. The resulting data mustthen be analyzed by scientists and agronomists to select an event thatis useful for commercial purposes. Once selected, such an event may thenbe introgressed using plant breeding methods as a single locus havingmultiple insect resistance traits into new germplasm suitably adapted tospecific local growing conditions, and stacked/combined by breeding withother different events conferring traits different from the traitsconferred by the event of the present invention.

SUMMARY OF THE INVENTION

The invention provides a novel transgenic corn event—MON 95379—thatprovides insecticidal control over Lepidopteran pests of corn. Theinvention also provides transgenic plants, plant cells, seeds, plantparts, and commodity products comprising event MON 95379. In anotherembodiment, the invention provides polynucleotides specific for eventMON 95379 and plants, plant cells, seeds, plant parts, progeny plants,and commodity products comprising event MON 95379. In yet anotherembodiment, methods related to event MON 95379 are provided.

Thus, in one aspect the invention provides a recombinant DNA moleculecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and acomplete complement thereof.

In one embodiment, the recombinant DNA molecule is derived from cornevent MON 95379 in a sample of seed containing said event having beendeposited as ATCC Accession No. PTA-125027.

Another aspect of the invention provides a DNA molecule comprising apolynucleotide segment of sufficient length to function as a DNA probethat hybridizes specifically under stringent hybridization conditionswith corn event MON 95379 DNA in a sample, wherein detectinghybridization of said DNA molecule under said stringent hybridizationconditions is diagnostic for the presence of corn event MON 95379 DNA insaid sample. In certain embodiments, the sample comprises a corn plant,corn plant cell, corn seed, corn plant part, corn progeny plant,processed corn seed, animal feed comprising corn, corn oil, corn meal,corn flour, corn flakes, corn bran, pasta made with corn, corn biomass,and fuel products produced using corn and corn parts.

Yet another aspect of the invention provides a pair of DNA molecules,comprising a first DNA molecule and a second DNA molecule different fromthe first DNA molecule, that function as DNA primers when used togetherin an amplification reaction with a sample containing corn event MON95379 template DNA to produce an amplicon diagnostic for the presence ofsaid corn event MON 95379 DNA in said sample, wherein said ampliconcomprises the nucleotide sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

Another embodiment of the invention is a method of detecting thepresence of a DNA segment diagnostic for corn event MON 95379 DNA in asample, said method comprising: a) contacting the sample with a DNAmolecule that functions as a probe and hybridizes specifically understringent conditions with corn event MON 95379; b) subjecting saidsample and said DNA molecule to stringent hybridization conditions; andc) detecting hybridization of said DNA molecule to said DNA in saidsample, wherein said detection is diagnostic for the presence of saidcorn event MON 95379 DNA in said sample.

Yet another embodiment of the invention is a method of detecting thepresence of a DNA segment diagnostic for corn event MON 95379 DNA in asample, the method comprising: a) contacting said sample with the pairof DNA molecules of the invention; b) performing an amplificationreaction sufficient to produce a DNA amplicon; and c) detecting thepresence of said DNA amplicon in said reaction, wherein said DNAamplicon comprises the nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ IDNO:10.

Another embodiment of the invention is a corn plant, corn plant part,corn cell, or part thereof comprising a recombinant polynucleotidemolecule comprising the nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ IDNO:10. This corn plant, corn plant part, corn cell, or part thereof isinsecticidal when provided in the diet of a Lepidopteran insect pest.Lepidopteran pests can include Fall Armyworm (Spodoptera frugiperda),Corn Earworm (Helicoverpa zea), Southwestern Corn Borer (Diatraeagrandiosella), Surgarcane Borer (Diatraea saccharalis), and LesserCornstalk Borer (Elasmopalpus lignosellus). In addition, the corn plantcan be further defined as progeny of any generation of a corn plantcomprising the corn event MON 95379.

Yet another embodiment of the invention is a method for protecting acorn plant from insect infestation, wherein said method comprisesproviding in the diet of a Lepidopteran insect pest an insecticidallyeffective amount of cells or tissue of a corn plant comprising cornevent MON 95379. Again, contemplated Lepidopteran insect pests includeFall Armyworm (Spodoptera frugiperda), Corn Earworm (Helicoverpa zea),Southwestern Corn Borer (Diatraea grandiosella), Surgarcane Borer(Diatraea saccharalis), and Lesser Cornstalk Borer (Elasmopalpuslignosellus).

Another embodiment of the invention is a method of producing an insectresistant corn plant comprising: a) sexually crossing two different cornplants with at least one of the two different corn plants comprisingtransgenic corn event MON 95379 DNA; b) sampling seed or tissue from theprogeny of step (a); c) detecting in said sample from step (b) progenycomprising corn event MON 95379 DNA; and d) selecting said progenycomprising corn event MON 95379 DNA.

A further embodiment of the invention is a corn seed, nonliving cornplant material, or a microorganism comprising a detectable amount of thenucleotide sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or completecomplements thereof.

Yet another embodiment of the invention is a commodity productcomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, orcomplete complements thereof. Contemplated commodity products includewhole or processed corn seed, animal feed comprising corn, corn oil,corn meal, corn flour, corn flakes, corn bran, corn biomass, and fuelproducts produced using corn and corn parts.

Another embodiment of the invention is a corn plant, corn plant part, orcorn seed thereof comprising DNA functional as a template when tested inDNA amplification method producing an amplicon diagnostic for thepresence of event MON 95379 DNA.

Yet another embodiment of the invention is a method of determining thezygosity of a corn plant or corn seed comprising event MON 95379comprising: a) contacting a sample comprising corn DNA with a primerpair that is capable of producing an amplicon of one of the toxin codingsequences encoding Cry1B.868 or Cry1Da_7; b) contacting said samplecomprising corn DNA with a primer pair that is capable of producing anamplicon of an internal standard known to be single-copy and homozygousin the corn plant; c) contacting the DNA sample with a probe set whichcontains at least a first probe that specifically hybridizes to one ofthe toxin coding sequences encoding Cry1B.868 or Cry1Da_7, and a secondprobe that specifically hybridizes to the internal standard genomic DNAknown to be single-copy and homozygous in the corn plant; d) performinga DNA amplification reaction using real-time PCR and determining thecycle thresholds (Ct values) of the amplicon corresponding to the toxincoding sequence and the single-copy, homozygous internal standard; e)calculating the difference (ΔCt) between the Ct value of thesingle-copy, homozygous internal standard amplicon and the Ct value ofthe toxin coding sequence amplicon; and f) determining zygosity, whereina ΔCt of about zero (0) indicates homozygosity of the inserted T-DNA ofevent MON 95739 and a ΔCt of about one (1) indicates heterozygosity ofthe inserted T-DNA of event MON 95379. In certain embodiments of thismethod, the primer pairs are selected from the group consisting of SEQID NO:18 combined with SEQ ID NO:19, and SEQ ID NO:21 combined with SEQID NO:22; and the probes are SEQ ID NO:20 and SEQ ID NO:23. In anotherembodiment, the primer pairs are selected from the group consisting ofSEQ ID NO:18 combined with SEQ ID NO:19, and SEQ ID NO:24 combined withSEQ ID NO:25; and the probes are SEQ ID NO:20 and SEQ ID NO:26. In yetanother embodiment of this invention the ΔCt of about one (1) indicatingheterozygosity of the inserted T-DNA of event MON 95379 is in the rangeof 0.75 to 1.25.

A further embodiment of the invention is a method of determining thezygosity of a corn plant or corn seed comprising event MON 95379comprising: a) contacting a sample comprising corn DNA with a set ofprimer pairs comprising at least two different primer pairs capable ofproducing a first amplicon diagnostic for event MON 95379 and a secondamplicon diagnostic for native corn genomic DNA not comprising event MON95379; i) performing a nucleic acid amplification reaction with thesample and the set of primer pairs; ii) detecting in the nucleic acidamplification reaction the first amplicon diagnostic for event MON95379, or the second amplicon diagnostic for native corn genomic DNA notcomprising event MON 95379, wherein the presence of only the firstamplicon is diagnostic of a corn plant or corn seed homozygous for eventMON 95379, and the presence of both the first amplicon and the secondamplicon is diagnostic of a corn plant or corn seed heterozygous forevent MON 95379; or b) contacting a sample comprising corn DNA with aprobe set which contains at least a first probe that specificallyhybridizes to event MON 95379 DNA and at least a second probe thatspecifically hybridizes to corn genomic DNA that was disrupted byinsertion of the heterologous DNA of event MON 95379 and does nothybridize to event MON 95379 DNA; i) hybridizing the probe set with thesample under stringent hybridization conditions, wherein detectinghybridization of only the first probe under the hybridization conditionsis diagnostic for a corn plant or corn seed homozygous for event MON95379, and wherein detecting hybridization of both the first probe andthe second probe under the hybridization conditions is diagnostic for acorn plant or a corn seed heterozygous for event MON 95379. In oneembodiment of this method, the set of primer pairs comprises SEQ IDNO:15 combined with SEQ ID NO:16, and SEQ ID NO:15 combined with SEQ IDNO:27. In another embodiment of this method, the probe set comprises SEQID NO:17 and SEQ ID NO:28.

The forgoing and other aspects of the invention will become moreapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the sequence of corn event MON 95379. Horizontal linesand boxes correspond to the positions of SEQ ID NO:1 ([1]), SEQ ID NO:2([2]), SEQ ID NO:3 ([3]), SEQ ID NO:4 ([4]), SEQ ID NO:5 ([5]), SEQ IDNO:6 ([6]), SEQ ID NO:7 ([7]), SEQ ID NO:8 ([8]), SEQ ID NO:9 ([9]), SEQID NO:11 ([11]), and SEQ ID NO:12 ([12]) relative to SEQ ID NO:10([10]). The horizontal arrows labeled SQ51219 (SEQ ID NO:15) ([15]),SQ21524 (SEQ ID NO:16) ([16]), SQ50998 (SEQ ID NO:21) ([21]), SQ50997(SEQ ID NO:22) ([22]), SQ50485 (SEQ ID NO:24) ([24]) and SQ50484 (SEQ IDNO:25) ([25]) represent the approximate position of subsets of primersthat can be used to detect corn event MON 95379. The horizontal arrowslabeled PB10269 (SEQ ID NO:17) ([17]), PB50340 (SEQ ID NO:23) ([23]),PB50138 (SEQ ID NO:26) ([26]) represent the approximate position of aDNA probe that can be used to detect corn event MON 95379. “E”represents an enhancer element, “P” represents a promoter element, “L”represents a leader (5′ UTR) element, “I” represents an intron element,“T” represents a 3′ UTR, “Cry1B.868” represents the Cry1B.868 codingsequence element, “Cry1Da_7” represents the Cry1Da_7 coding sequenceelement, “LoxP” represents the site at which Cre-recombinase markerexcision occurred, leaving behind one of the two LoxP sites after markerexcision, and “LB” represents the left T-DNA border.

FIG. 2 is a diagrammatic representation of the T-DNA cassettes beforeintegration to form event MON 95379, after integration, and afterCre-excision. The top horizontal box represents the T-DNA cassette inthe plasmid vector used to transform event MON 95379, presented as SEQID NO:13 ([13]) (“T-DNA Before Integration”). The horizontal arrowsbelow [13] represent the individual genetic elements comprised withinthe two transgene cassettes. “LB” represents a T-DNA left borderelement, “E” represents an enhancer element, “P” represents a promoterelement, “L” represents a leader (5′ UTR) element, “I” represents anintron element, “T” represents a 3′ UTR, “Cry1B.868” represents theCry1B.868 coding sequence element, “Cry1Da_7” represents the Cry1Da_7coding sequence element, “CP4” represents the CP4 selectable marker,“TS” represented a targeting sequence, “LoxP” represents the site atwhich Cre-recombinase marker excision occurs, and “RB” represents aT-DNA right border element. The middle horizontal box, “Inserted T-DNAAfter Integration,” represents the T-DNA cassette integrated into thecorn genome after transformation wherein the right T-DNA border (RB) waslost during integration. The bottom horizontal box, “Inserted T-DNAAfter Cre-Excision,” represents the integrated T-DNA cassette after theCP4 selectable marker cassette was excised, leaving behind one of thetwo LoxP sites and the LB region.

FIG. 3 is a diagrammatic representation of the breeding process toproduce the marker-free corn event MON 95379. R₀ generation events(“transformants”) are those that are derived from the initialtransformation with the binary transformation vector used to generatecorn event MON 95379. Subsequent “R” generations (R₁, and R₂) representsuccessive generations produced through self-pollination of plantsderived from the initial R₀ transformant that resulted in the corn eventMON 95379. The R₂ transformants which are homozygous for the T-DNAinsertion are cross-pollinated with an elite transgenic corn linecomprising a transgene cassette for the expression of Cre-recombinase,resulting in an F1 generation, wherein many of the progeny have lost theCP4 selectable marker cassette due to Cre-recombinase excision.Hemizygous T-DNA positive, CP4 negative plants are selected andself-pollinated, resulting in an F2 generation. F2 plants homozygous forthe inserted T-DNA allele without the CP4 marker and lacking theCre-recombinase transgene cassette are selected and self-pollinatedgiving rise to an F3 generation. The F₃ generation plants areself-pollinated giving rise to a pure line of F₄ Gold Standard Seed.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a 50 nucleotide sequence representing the 5′ junctionregion of corn genomic DNA and the integrated transgenic expressioncassette. SEQ ID NO:1 is found within SEQ ID NO:10 at nucleotidepositions 838-887.

SEQ ID NO:2 is a 50 nucleotide sequence representing the 3′ junctionregion of the integrated transgenic expression cassette and the corngenomic DNA. SEQ ID NO:2 is found within SEQ ID NO:10 at nucleotidepositions 14,156-14,205.

SEQ ID NO:3 is a 100 nucleotide sequence representing the 5′ junctionregion of corn genomic DNA and the integrated transgenic expressioncassette. SEQ ID NO:3 is found within SEQ ID NO:10 at nucleotidepositions 813-912.

SEQ ID NO:4 is a 100 nucleotide sequence representing the 3′ junctionregion of the integrated transgenic expression cassette and the corngenomic DNA. SEQ ID NO:4 is found within SEQ ID NO:10 at nucleotidepositions 14,131-14,230.

SEQ ID NO:5 is a 200 nucleotide sequence representing the 5′ junctionregion of corn genomic DNA and the integrated transgenic expressioncassette. SEQ ID NO:5 is found within SEQ ID NO:10 at nucleotidepositions 763-962.

SEQ ID NO:6 is a 200 nucleotide sequence representing the 3′ junctionregion of the integrated transgenic expression cassette and the corngenomic DNA. SEQ ID NO:6 is found within SEQ ID NO:10 at nucleotidepositions 14,081-14,280.

SEQ ID NO:7 is a 1.160 nucleotide sequence representing the 5′ junctionregion of corn genomic DNA and the integrated transgenic expressioncassette. SEQ ID NO:7 is found within SEQ ID NO:10 at nucleotidepositions 1-1,160.

SEQ ID NO:8 is a 1,178 nucleotide sequence representing the 3′ junctionregion of the integrated transgenic expression cassette and the corngenomic DNA. SEQ ID NO:8 is found within SEQ ID NO:10 at nucleotidepositions 14,039-15,216.

SEQ ID NO:9 is a 13,318 nucleotide sequence corresponding to thetransgenic inserted T-DNA of corn event MON 95379.

SEQ ID NO:10 is a 15.216 nucleotide sequence corresponding to the contignucleotide sequence of the 5′ genomic flanking DNA nucleotide sequence,the inserted T-DNA nucleotide sequence in event MON 95379, and the 3′genomic flanking DNA nucleotide sequence; and includes SEQ ID NO:11(nucleotides 1-862), SEQ ID NO:9 (nucleotides 863-14,180), and SEQ IDNO:12 (nucleotides 14,181-15,216).

SEQ ID NO:11 is an 862 nucleotide sequence representing the 5′ flankingcorn genomic DNA up to the inserted T-DNA. SEQ ID NO:11 is found withinSEQ ID NO:10 at nucleotide positions 1-862.

SEQ ID NO:12 is a 1,036 nucleotide sequence representing the 3′ flankingcorn genomic DNA after the inserted T-DNA. SEQ ID NO:12 is found withinSEQ ID NO:10 at nucleotide positions 14,181-15,216.

SEQ ID NO:13 is a 18,376 nucleotide sequence representing the transgenecassette comprised within the binary plasmid transformation vector usedto transform corn to produce corn event MON 95379.

SEQ ID NO:14 is a 35 nucleotide sequence representing the LoxP sitesused for Cre-mediated excision and recombination. A remaining LoxP siteafter marker excision can be found within SEQ ID NO:10 at nucleotidepositions 1,080-1,114.

SEQ ID NO:15 is a 20 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ51219 used to identify corn eventMON 95379 DNA in a sample, and is identical to the nucleotide sequencecorresponding to positions 833-852 of SEQ ID NO:10.

SEQ ID NO:16 is a 30 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ21524 used to identify corn eventMON 95379 DNA in a sample, and is identical to the reverse complement ofthe nucleotide sequence corresponding to positions 905-934 of SEQ IDNO:10.

SEQ ID NO:17 is a 16 nucleotide sequence corresponding to a probereferred to as PB10269 used to identify corn event MON 95379 DNA in asample, and is identical to the reverse complement of the nucleotidesequence corresponding to positions 886-901 of SEQ ID NO:10.

SEQ ID NO:18 is a 24 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ20222 used as an internal controlfor the event and zygosity assay for MON 95379 and hybridizes to aregion of the corn genome.

SEQ ID NO:19 is a 28 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ20221 used as an internal controlfor the event and zygosity assay for MON 95379 and hybridizes to aregion of the corn genome.

SEQ ID NO:20 is a 29 nucleotide sequence corresponding to a probereferred to as PB50237 used as an internal control for the event andzygosity assay for MON 95379 and hybridizes to a region of the corngenome.

SEQ ID NO:21 is a 20 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ50998 used in the zygosity assayfor event MON 95379 and hybridizes to the coding sequence of Cry1B.868within SEQ ID NO:10; and is identical to the nucleotide sequencecorresponding to positions 2,809-2,828 of SEQ ID NO:10.

SEQ ID NO:22 is a 20 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ50997 used in the zygosity assayfor event MON 95379 and hybridizes to the coding sequence of Cry1B.868within SEQ ID NO:10; and is identical to the reverse complement of thenucleotide sequence corresponding to positions 2,852-2,871 of SEQ IDNO:10.

SEQ ID NO:23 is an 18 nucleotide sequence corresponding to a probereferred to as PB50340 used in the zygosity assay for event MON 95379and hybridizes to the coding sequence of Cry1B.868 within SEQ ID NO:10;and is identical to the reverse complement of the nucleotide sequencecorresponding to positions 2,833-2,850 of SEQ ID NO:10.

SEQ ID NO:24 is a 19 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ50485 used in the zygosity assayfor event MON 95379 and hybridizes to the coding sequence of Cry1Da_7within SEQ ID NO:10; and is identical to the nucleotide sequencecorresponding to positions 12,820-12,838 of SEQ ID NO:10.

SEQ ID NO:25 is an 18 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ50484 used in the zygosity assayfor event MON 95379 and hybridizes to the coding sequence of Cry1Da_7within SEQ ID NO:10; and is identical to the reverse complement of thenucleotide sequence corresponding to positions 12,855-12,872 of SEQ IDNO:10.

SEQ ID NO:26 is a 14 nucleotide sequence corresponding to a probereferred to as PB50138 used in the zygosity assay for event MON 95379and hybridizes to the coding sequence of Cry1Da_7 within SEQ ID NO:10;and is identical to the reverse complement of the nucleotide sequencecorresponding to positions 12,840-12,853 of SEQ ID NO:10.

SEQ ID NO:27 is a 21 nucleotide sequence corresponding to a thermalamplification primer referred to as PNEGDNA used in the zygosity assayfor event MON 95379 and hybridizes to a region of corn genomic DNA whichwas deleted when the T-DNA used to produce event MON 95379 inserted intothe corn genome. An amplicon derived from the combination of primersSQ51219 and PNEGDNA is diagnostic for the wild-type allele lacking theevent MON 95379 inserted T-DNA.

SEQ ID NO:28 is a 14 nucleotide sequence corresponding to a probereferred to as PRBNEGDNA used in the zygosity assay for event MON 95379and hybridizes to a region of corn genomic DNA which was deleted whenthe T-DNA used to produce event MON 95379 inserted into the corn genome.

DETAILED DESCRIPTION

The present invention provides a transgenic corn event—MON 95379—thatachieves insecticidal control over Lepidopteran pests of corn byexpression of Cry1B.868 and Cry1Da_7. Specifically, expression of theCry1B.868 and Cry1Da_7 insect inhibitory proteins in corn event MON95379 provides resistance to the Lepidopteran insect pests Fall Armyworm(Spodoptera frugiperda), Corn Earworm (Helicoverpa zea), SouthwesternCorn Borer (Diatraea grandiosella), Surgarcane Borer (Diatraeasaccharalis), and Lesser Cornstalk Borer (Elasmopalpus lignosellus).Event MON 95379 will meet a great need for control of these insects inthe corn market, as chemical insecticides often do not provide adequatecontrol of these insects, or require multiple applications over thegrowing season, increasing the input of chemical pesticides in theenvironment and adding cost to the production of corn.

It should be understood that reference to event MON 95379 is equivalentto reference to event MON95379; they are interchangeable and representthe same transgenic corn event.

Plant transformation techniques are used to insert foreign DNA (alsoknown as transgenic DNA) randomly into a chromosome of the genome of acell to produce a genetically engineered cell, also referred to as a“transgenic” or “recombinant” cell. Using this technique, manyindividual cells are transformed, each resulting in a unique “transgenicevent” or “event” due to the random insertion of the foreign DNA intothe genome. A transgenic plant is then regenerated from each individualtransgenic cell. This results in every cell of the transgenic plantcontaining the uniquely inserted transgenic event as a stable part ofits genome. This transgenic plant can then be used to produce progenyplants, each containing the unique transgenic event.

Corn event MON 95379 was produced by an Agrobacterium-mediatedtransformation process of corn immature embryos with a single T-DNAbinary system. In this system, an Agrobacterium strain employing onebinary plasmid vector with a single T-DNA was utilized. The T-DNAconstruct comprised two transgene cassettes for the expression of theinsect toxin coding sequences encoding Cry1B.868 and Cry1Da_7, and atransgene cassette used for the selection of transformed corn cellsusing glyphosate selection (CP4). The T-DNA construct is SEQ ID NO:13,and illustrated in FIG. 2 (“T-DNA Before Integration”). Duringintegration, the right T-DNA border was lost as shown in FIG. 2(“Inserted T-DNA After Integration”). The glyphosate selection cassettewas flanked on both sides with LoxP recognition sites which arerecognized by Cre-recombinase, derived from Enterobacteria phage P1(Larry Gilbertson (2003) Cre-lox recombination: Cre-active tools forplant biotechnology. TRENDS in Biotechnology. 21:12, 550-555).

As specifically described herein, corn event MON 95379 was produced by acomplex research and development process in which: (1) hundreds ofplasmid vector constructs—which varied with respect to the codingsequences for the insecticidal proteins, the coding sequences for thetranscriptional regulatory elements, and number and orientation of thecassettes within the constructs—were developed and transformed into corncells to create thousands of events that were tested and analyzed,resulting in the selection of the construct used to generate event MON95379; (2) hundreds of corn cells were transformed with the constructused to generate event MON 95379, creating a population of transgenicplants in which each plant contained a unique transgenic event that wasregenerated and tested; (3) the final event MON 95379 was selected aftera rigorous multi-year event selection process involving the testing andanalysis of molecular characteristics, efficacy, protein expression, andagronomic properties in a variety of genetic backgrounds; and (4) theglyphosate selection cassette in corn event MON 95379 was removedthrough in vivo Cre-excision to create a “marker-free” final event MON95379. Corn event MON 95379 was thus produced and selected as a uniquelysuperior event useful for broad-scale agronomic purposes.

The plasmid DNA inserted into the genome of corn event MON 95379 wascharacterized by detailed molecular analysis. This analysis included:the insert number (number of integration sites within the corn genome),the genomic insert location (the specific site in the corn genome wherethe insertion occurred), the copy number (the number of copies of theT-DNA within one locus), and the integrity of the transgenic insertedDNA. The detailed molecular analysis demonstrated that the integratedT-DNA containing the Cry1B.868 and Cry1Da_7 expression cassettesremained intact after integration and Cre-excision of the glyphosate(CP4) selection cassette. As used herein, an “expression cassette” or“cassette” is a recombinant DNA molecule comprising a combination ofdistinct elements that are to be expressed by a transformed cell. Table1 provides a list of the elements contained in SEQ ID NO:10, the DNAsequence that corresponds to corn event MON 95379.

TABLE 1 Description of corn event MON 95379 Position in SEQ ID ElementNO: 10 Description 5′ Flanking DNA  1-862 DNA sequence flanking the 5′end of the transgenic insert. Left Border Region  863-1044 DNA regionfrom Agrobacterium tumefaciens containing the left border sequence. LoxP1080-1114 A recognition sequence for a site-specific recombinase fromEnterobacteria phage P1. T-Os.LTP:1 1225-1524 The 3' untranslated regionfor a Lipid Transfer Protein-like gene (LTP) from Oryza sativa (rice).Cry1B.868 1534-5133 Coding sequence of a chimeric insect toxin comprisedof domains 1 and 2 of Cry1Be2, domain 3 of Cry1Ca, and the protoxindomain of Cry1Ab3. I-Zm.UbqM1-1:1:16 5160-6212 An intron derived from anUbquitin 1 gene of Zea mays Mexicana. L-Zm.UbqM1-1:1:4 6213-6290 A 5′UTR derived from an Ubquitin 1 gene of Zea mays Mexicana.P-Zm.UbqM1-1:1:5 6291-7167 A promoter derived from an Ubquitin 1 gene ofZea mays Mexicana. E-FMV.35S-1:1:2 7195-7731 The enhancer of the 35Sgene from Figwort Mosaic Virus (FMV). P-SETit.Tip-1:1:1 7743-8659 Apromoter of a tonoplast membrane integral protein gene from Setariaitalica (foxtail millet). L-SETit.Tip-1:1:1 8660-8723 A 5′ UTR of atonoplast membrane integral protein gene from Setaria italica (foxtailmillet). I-Os.Act15-1:1:1  8732-10024 The first intron from the Actin 15(Act 15) gene from Oryza sativa (rice). Cry1Da_7 10043-13543 Codingsequence of a Cry1Da insect toxin with amino acid modifications toimprove efficacy. T-Os.GOS2-1:1:1 13560-14027 The 3′ untranslated regionfrom the GOS2 gene encoding a translation initiation factor from Oryzasativa (rice). 3′ Flanking Sequence 14181-15216 DNA sequence flankingthe 3′ end of the transgenic insert.

Corn event MON 95379 is characterized as an insertion into a singlelocus in the corn genome, resulting in two new loci or junctionsequences (e.g., sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ IDNO:8) spanning a portion of the inserted DNA and the corn genomic DNAthat are not known to appear naturally in the corn genome or othertransgenic corn events—they are unique to event MON 95379. Thesejunction sequences are useful in detecting the presence of the event MON95379 in corn cells, corn tissue, corn seed, and corn plants or cornplant products, such as corn commodity products. DNA molecular probesand primer pairs are described herein that have been developed for usein identifying the presence of these various junction segments inbiological samples containing or suspected of containing corn cells,corn seed, corn plant parts, or corn plant tissue that contain the eventMON 95379.

A sample is intended to refer to a composition that is eithersubstantially pure corn DNA or a composition that contains corn DNA. Ineither case, the sample is a biological sample, i.e., it containsbiological materials, including but not limited to DNA obtained orderived from, either directly or indirectly, the genome of corn eventMON 95379. “Directly” refers to the ability of the skilled artisan todirectly obtain DNA from the corn genome by fracturing corn cells (or byobtaining samples of corn that contain fractured corn cells) andexposing the genomic DNA for the purposes of detection. “Indirectly”refers to the ability of the skilled artisan to obtain the target orspecific reference DNA, i.e., a novel and unique junction segmentdescribed herein as being diagnostic for the presence of the event MON95379 in a particular sample, by means other than by obtaining directlyvia fracturing of corn cells or obtaining a sample of corn that containsfractured corn cells. Such indirect means include, but are not limitedto, amplification of a DNA segment that contains the DNA sequencetargeted by a particular probe designed to bind with specificity to thetarget sequence, or amplification of a DNA segment that can be measuredand characterized, i.e., measured by separation from other segments ofDNA through some efficient matrix such as an agarose or acrylamide gelor the like, or characterized by direct sequence analysis of theamplicons, or cloning of the amplicon into a vector and directsequencing of the inserted amplicon present within such vector.

Detailed molecular analysis demonstrated that event MON 95379 contains asingle T-DNA insertion with one copy of each of the Cry1B.868 andCry1Da_7 expression cassettes. No additional elements from thetransformation construct other than portions of the Agrobacteriumtumefaciens left border region used for transgenic DNA transfer from theplant transformation plasmid to the corn genome were identified in eventMON 95379. Finally, thermal amplification producing specific ampliconsdiagnostic for the presence of event MON 95379 in a sample and DNAsequence analyses were performed to determine the arbitrarily assigned5′ and 3′ insert-to-plant genome junctions, confirm the organization ofthe elements within the insert, and determine the complete DNA sequenceof the inserted transgenic DNA (SEQ ID NO:9). SEQ ID NO:11 is a sequencerepresenting the eight hundred sixty-two (862) base-pair (bp) 5′ LH244corn genomic DNA sequence flanking the inserted T-DNA sequence presentedas SEQ ID NO:9. SEQ ID NO:12 is a sequence representing the one thousandthirty-six (1.036) bp 3′ LH244 corn genomic DNA sequence flanking theinserted T-DNA sequence presented as SEQ ID NO:9. SEQ ID NO:7 is asequence representing the eight hundred sixty-two (862) base-pair (bp)5′ LH244 corn genomic DNA sequence flanking the inserted T-DNA sequencecombined with two hundred ninety-eight (298) bp of inserted T-DNAsequence presented as SEQ ID NO:9. SEQ ID NO:8 is a sequencerepresenting one hundred forty-two (142) bp of inserted T-DNA sequencewith the one thousand thirty-six (1.036) bp 3′ LH244 corn genomic DNAsequence flanking the inserted T-DNA sequence presented as SEQ ID NO:9.SEQ ID NO:10 corresponds to corn event MON 95379 and contains acontiguous sequence (contig) comprising the 5′ LH244 flanking sequence,the transgene insert of event MON 95379, and the 3′ LH244 flankingsequence, and thus contains the insert-to-plant genome junctionsequences.

Unless otherwise noted herein, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art.Definitions of common terms in molecular biology may be found in Riegeret al., Glossary of Genetics: Classical and Molecular, 5^(th) edition,Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford UniversityPress: New York, 1994, along with other sources known to those ofordinary skill in the art. As used herein, the term “corn” means speciesbelong to the genus Zea, preferably Zea mays and includes all plantvarieties that can be bred with corn plants containing event MON 95379,including wild corn species as well as those plants belonging to thegenus Zea that permit breeding between species.

The present invention provides for transgenic plants which have beentransformed with a DNA construct that contains expression cassettesexpressing toxic amounts of the insecticidal proteins Cry1B.868 andCry1Da_7. What is meant by toxic amount is an efficacious amount, aninsecticidal amount, an insecticidally-effective amount, a target insectsuppressive amount, an efficacious pesticidal amount, an amount in thediet of insects in the order of Lepidoptera that is insecticidal, andother similar terms to be understood according to conventional usage bythose of ordinary skill in the relevant art. Corn plants transformedaccording to the methods and with the DNA construct disclosed herein areresistant to Lepidopteran insect pests.

A transgenic “plant” is produced by transformation of a plant cell withheterologous DNA, i.e., a polynucleic acid construct that includes anumber of efficacious features of interest, regeneration of a plantresulting from the insertion of the transgene into the genome of theplant cell, and selection of a particular plant characterized byinsertion into a particular genome location and the number ofefficacious features of the regenerated transgenic plant. The term“event” refers to DNA from the original transformant comprising theinserted DNA, and flanking genomic sequences immediately adjacent to theinserted DNA. Such DNA is unique and would be expected to be transferredto a progeny that receives the inserted DNA, including the transgene ofinterest, as the result of a sexual cross of a parental line thatincludes the inserted DNA (e.g., the original transformant and progenyresulting from selfing) and a parental line that does not contain theinserted DNA. The present invention also provides the originaltransformant plant and progeny of the transformant that include theheterologous DNA. Such progeny may be produced by a sexual outcrossbetween plants comprising the event and another plant wherein theprogeny includes the heterologous DNA. Even after repeated back-crossingto a recurrent parent, the event is present in the progeny of the crossat the same chromosomal location.

As used herein, the term “recombinant” refers to a non-natural DNA,protein, or organism that would not normally be found in nature and wascreated by human intervention. A “recombinant DNA molecule” is a DNAmolecule comprising a combination of DNA molecules that would notnaturally occur together and is the result of human intervention. Forexample, a DNA molecule that is comprised of a combination of at leasttwo DNA molecules heterologous to each other, such as a DNA moleculethat comprises a transgene and the plant genomic DNA adjacent to thetransgene, is a recombinant DNA molecule.

The terms “DNA” and “DNA molecule” referred to herein refer to adeoxyribonucleic acid (DNA) molecule. A DNA molecule may be of genomicor synthetic origin, and is by convention from the 5′ (upstream) end tothe 3′ (downstream) end. As used herein, the term “DNA sequence” refersto the nucleotide sequence of the DNA molecule. By convention, the DNAsequences of the invention and fragments thereof are disclosed withreference to only one strand of the two strand complementary DNAsequence strands. By implication and intent, the complementary sequencesof the sequences provided here (the sequences of the complementarystrand), also referred to in the art as the reverse complementarysequences, are within the scope of the invention and are expresslyintended to be within the scope of the subject matter claimed.

As used herein, the term “fragment” refers to a smaller piece of thewhole. For example, fragments of SEQ ID NO:10 would include sequencesthat are at least about 12 consecutive nucleotides, at least about 13consecutive nucleotides, at least about 14 consecutive nucleotides, atleast about 15 consecutive nucleotides, at least about 16 consecutivenucleotides, at least about 17 consecutive nucleotides, at least about18 consecutive nucleotides, at least about 19 consecutive nucleotides,at least about 20 consecutive nucleotides, at least about 25 consecutivenucleotides, at least about 30 consecutive nucleotides, at least about35 consecutive nucleotides, at least about 40 consecutive nucleotides,at least about 45 consecutive nucleotides, at least about 50 consecutivenucleotides, at least about 60 consecutive nucleotides, at least about70 consecutive nucleotides, at least about 80 consecutive nucleotides,at least about 90 consecutive nucleotides, or at least about 100consecutive nucleotides of the complete sequence of SEQ ID NO:10.

Reference in this application to an “isolated DNA molecule” or anequivalent term or phrase is intended to mean that the DNA molecule isone that is present alone or in combination with other compositions, butnot within its natural environment. For example, nucleic acid elementssuch as a coding sequence, intron sequence, untranslated leadersequence, promoter sequence, transcriptional termination sequence, andthe like, that are naturally found within the DNA of the genome of anorganism are not considered to be “isolated” so long as the element iswithin the genome of the organism and at the location within the genomein which it is naturally found. However, each of these elements, andsubparts of these elements, would be “isolated” within the scope of thisdisclosure so long as the element is not within the genome of theorganism and at the location within the genome in which it is naturallyfound. Similarly, a nucleotide sequence encoding an insecticidal proteinor any naturally occurring insecticidal variant of that protein would bean isolated nucleotide sequence so long as the nucleotide sequence wasnot within the DNA of the bacterium from which the sequence encoding theprotein is naturally found. A synthetic nucleotide sequence encoding theamino acid sequence of the naturally occurring insecticidal proteinwould be considered to be isolated for the purposes of this disclosure.For the purposes of this disclosure, any transgenic nucleotide sequence,i.e., the nucleotide sequence of the DNA inserted into the genome of thecells of a plant or bacterium, or present in an extrachromosomal vector,would be considered to be an isolated nucleotide sequence whether it ispresent within the plasmid or similar structure used to transform thecells, within the genome of the plant or bacterium, or present indetectable amounts in tissues, progeny, biological samples or commodityproducts derived from the plant or bacterium. In any circumstance, theisolated DNA molecule is a chemical molecule, regardless of whether itis referred to as a nucleic acid, a nucleic acid sequence, apolynucleotide sequence, and the like. It is a novel, inventive moleculethat exhibits industrial applicability both when present in a plant cellor in a plant genome, and when present outside of a plant cell, andtherefore, exhibits and is intended to exhibit such utility regardlessof where the molecule is located.

The DNA sequence of the region spanning the connection by phosphodiesterbond linkage of one end of the transgenic insert to the flanking corngenomic DNA is referred to as a “junction.” A junction is the connectionpoint of the transgenic insert and flanking DNA as one contiguousmolecule. One junction is found at the 5′ end of the transgenic insertand the other is found at the 3′ end of the transgenic insert, referredto herein as the 5′ and 3′ junction, respectively. A “junction sequence”refers to a DNA sequence of any length that spans the 5′ or 3′ junctionof an event. Junction sequences of corn event MON 95379 are apparent toone of skill in the art using SEQ ID NO:10. Examples of junctionsequences of event MON 95379 are provided as SEQ ID NOs:1-8. FIG. 1illustrates the physical arrangement of the junction sequences, arrangedfrom 5′ to 3′, relative to SEQ ID NO:10. The junction sequences of eventMON 95379 may be present as part of the genome of a plant, seed, or cellcontaining event MON 95379. The identification of any one or more of thejunction sequences in a sample from a plant, plant part, seed, or cellindicates that the DNA was obtained from corn containing event MON95379, and is diagnostic for the presence of event MON 95379.

The junction sequences for event MON 95379 may be represented by asequence from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,and SEQ ID NO:10. For example, the junction sequences may be arbitrarilyrepresented by the nucleotide sequences provided as SEQ ID NO:1 and SEQID NO:2. Alternatively, the junction sequences may be arbitrarilyrepresented by the nucleotide sequences provided as SEQ ID NO:3 and SEQID NO:4. Alternatively, the junction sequences may be arbitrarilyrepresented by the nucleotide sequences provided as SEQ ID NO:5 and SEQID NO:6. Alternatively, the junction sequences may be arbitrarilyrepresented by the nucleotide sequences provided as SEQ ID NO:7 and SEQID NO:8. These nucleotides are connected by phosphodiester linkage, andin corn event MON 95379 are present as part of the recombinant plantcell genome.

The identification of one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,and SEQ ID NO:10 in a sample derived from a corn plant, corn seed, orcorn plant part is diagnostic that the DNA was obtained from corn eventMON 95379. The invention thus provides a DNA molecule that contains atleast one of the nucleotide sequences provided as SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Any segment of DNA derivedfrom transgenic corn event MON 95379 that is sufficient to include atleast one of the sequences provided as SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, and SEQ ID NO:10 is within the scope of the invention. Inaddition, any polynucleotide comprising a sequence complementary to anyof the sequences described within this paragraph is within the scope ofthe invention.

The invention provides exemplary DNA molecules that can be used eitheras primers or probes for detecting the presence of DNA derived from acorn plant comprising event MON 95379 DNA in a sample. Such primers orprobes are specific for a target nucleic acid sequence and, as such, areuseful for the identification of corn event MON 95379 nucleic acidsequence by the methods of the invention described herein.

A “probe” is a nucleic acid molecule that is complementary to a strandof target nucleic acid and is useful in hybridization methods. A probemay be attached a conventional detectable label or reporter molecule,e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme.Such a probe is complementary to a strand of a target nucleic acid and,in the case of the present invention, to a strand of DNA from event MON95379 whether from an event MON 95379 containing plant or from a samplethat includes event MON 95379 DNA. Probes according to the presentinvention include not only deoxyribonucleic or ribonucleic acids, butalso polyamides and other probe materials that bind specifically to atarget DNA sequence and can be used to detect the presence of thattarget DNA sequence. Exemplary DNA sequences useful as a probe fordetecting corn event MON 95379 are provided as: SEQ ID NO:17 (PB10269),SEQ ID NO:23 (PB50340); SEQ ID NO:26 (PB50138).

A “primer” is typically a DNA molecule that is designed for use inspecific annealing or hybridization methods that involve thermalamplification. A pair of primers may be used with template DNA (such asa sample of corn genomic DNA) in a thermal amplification (such aspolymerase chain reaction (PCR)) to produce an amplicon, where theamplicon produced from such reaction would have a DNA sequencecorresponding to sequence of the template DNA located between the twosites where the primers hybridized to the template.

A primer is typically designed to hybridize to a complementary targetDNA strand to form a hybrid between the primer and target DNA strand,and the presence of the primer is a point of recognition by a polymeraseto begin extension of the primer (i.e., polymerization of additionalnucleotides into a lengthening nucleotide molecule) using as a templatethe target DNA strand. Primer pairs refer to use of two primers bindingopposite strands of a double stranded nucleotide segment for the purposeof amplifying linearly the polynucleotide segment between the positionstargeted for binding by the individual members of the primer pair,typically in a thermal amplification reaction or other conventionalnucleic-acid amplification methods. Exemplary DNA molecules useful asprimers are provided as SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25 and SEQ IDNO:27.

The primer pair SEQ ID NO:15 and SEQ ID NO:16 are useful as a first DNAmolecule and a second DNA molecule that is different from the first DNAmolecule, and both are each of sufficient length of contiguousnucleotides of SEQ ID NO:10 to function as DNA primers that, when usedtogether in a thermal amplification reaction with template DNA derivedfrom corn event MON 95379, to produce an amplicon diagnostic for cornevent MON 95379 DNA in a sample. The primer pair SEQ ID NO:21 and SEQ IDNO:22 are useful as a first DNA molecule and a second DNA molecule thatis different from the first DNA molecule, and both are each ofsufficient length of contiguous nucleotides of SEQ ID NO:10 to functionas DNA primers that, when used together in a thermal amplificationreaction with template DNA derived from corn event MON 95379, to producean amplicon diagnostic for the zygosity of corn event MON 95379 DNA in asample. The primer pair SEQ ID NO:24 and SEQ ID NO:25 are useful as afirst DNA molecule and a second DNA molecule that is different from thefirst DNA molecule, and both are each of sufficient length of contiguousnucleotides of SEQ ID NO:10 to function as DNA primers that, when usedtogether in a thermal amplification reaction with template DNA derivedfrom corn event MON 95379, to produce an amplicon diagnostic for thezygosity of corn event MON 95379 DNA in a sample. The primer pair SEQ IDNO:18 and SEQ ID NO:19 are useful as a first DNA molecule and a secondDNA molecule that is different from the first DNA molecule, and both areeach of sufficient length of contiguous nucleotides of a locus withinthe corn genome to function as DNA primers that, when used together in athermal amplification reaction with template DNA derived from corn eventMON 95379, to produce an amplicon that serves as an internal control forboth the diagnosis of corn event MON 95379, as well as the zygosity ofcorn event MON 95379 DNA in a sample.

DNA probes and DNA primers are generally eleven (11) polynucleotides ormore in length, often eighteen (18) polynucleotides or more, twenty-four(24) polynucleotides or more, or thirty (30) polynucleotides or more.Such probes and primers are selected to be of sufficient length tohybridize specifically to a target sequence under high stringencyhybridization conditions. Preferably, probes and primers according tothe present invention have complete sequence similarity with the targetsequence, although probes differing from the target sequence that retainthe ability to hybridize to target sequences may be designed byconventional methods.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA molecule. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic plant in a sample.Polynucleic acid molecules also referred to as nucleic acid segments orfragments thereof are capable of specifically hybridizing to othernucleic acid molecules under certain circumstances.

As used herein, two polynucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure. Anucleic acid molecule is said to be the “complement” of another nucleicacid molecule if they exhibit complete complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to be“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989, and by Haymes et al.,In: Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, DC (1985). Departures from complete complementarity aretherefore permissible, as long as such departures do not completelypreclude the capacity of the molecules to form a double-strandedstructure. In order for a nucleic acid molecule to serve as a primer orprobe it need only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. Appropriate stringency conditions that promoteDNA hybridization, for example, 6.0×sodium chloride/sodium citrate (SSC)at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In a preferredembodiment, a polynucleic acid of the present invention willspecifically hybridize to one or more of the nucleic acid molecules setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ IDNO:10, or complements thereof or fragments thereof under moderatelystringent conditions, for example at about 2.0×SSC and about 65° C. In aparticularly preferred embodiment, a nucleic acid of the presentinvention will specifically hybridize to one or more of the nucleic acidmolecules set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,or SEQ ID NO:10, or complements or fragments thereof under highstringency conditions. In one aspect of the present invention, apreferred marker nucleic acid molecule of the present invention has thenucleic acid sequence set forth in SEQ ID NO:1, or SEQ ID NO:2, or SEQID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:6, or SEQ ID NO:7,or SEQ ID NO:8, or SEQ ID NO:9, or SEQ ID NO:10, or complements thereof,or fragments thereof. The hybridization of the probe to the target DNAmolecule can be detected by any number of methods known to those skilledin the art, these can include, but are not limited to, fluorescent tags,radioactive tags, antibody based tags, and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofpolynucleic acid amplification method directed to a target polynucleicacid molecule that is part of a polynucleic acid template. For example,to determine whether a corn plant resulting from a sexual cross containstransgenic plant genomic DNA from a corn plant comprising event MON95379 of the present invention. DNA that is extracted from a corn planttissue sample may be subjected to a polynucleic acid amplificationmethod using a primer pair that includes a first primer derived from agenomic DNA sequence in the region flanking the heterologous insertedDNA of event MON 95379 and is elongated by polymerase 5′ to 3′ in thedirection of the inserted DNA. The second primer is derived from theheterologous inserted DNA molecule is elongated by the polymerase 5′ to3′ in the direction of the flanking genomic DNA from which the firstprimer is derived. The amplicon may range in length from the combinedlength of the primer pair plus one nucleotide base pair, or plus aboutfifty nucleotide base pairs, or plus about two hundred-fifty nucleotidebase pairs, or plus about four hundred-fifty nucleotide base pairs ormore. Alternatively, a primer pair can be derived from genomic sequenceon both sides of the inserted heterologous DNA so as to produce anamplicon that includes the entire insert polynucleotide sequence (e.g.,a forward primer isolated from the genomic portion on the 5′ end of SEQID NO:10 and a reverse primer isolated from the genomic portion on the3′ end of SEQ ID NO:10 that amplifies a DNA molecule comprising theinserted DNA sequence (SEQ ID NO:9) identified herein in the event MON95379 genome). A member of a primer pair derived from the plant genomicsequence adjacent to the inserted transgenic DNA is located a distancefrom the inserted DNA sequence, this distance can range from onenucleotide base pair up to about twenty thousand nucleotide base pairs.The use of the term “amplicon” specifically excludes primer dimers thatmay be formed in the DNA thermal amplification reaction.

For practical purposes, one should design primers which produceamplicons of a limited size range, for example, between 100 to 1000bases. Smaller (shorter polynucleotide length) sized amplicons ingeneral are more reliably produced in thermal amplification reactions,allow for shorter cycle times, and can be easily separated andvisualized on agarose gels or adapted for use in endpoint TAQMAN®-likeassays. Smaller amplicons can be produced and detected by methods knownin the art of DNA amplicon detection. In addition, amplicons producedusing the primer pairs can be cloned into vectors, propagated, isolated,and sequenced or can be sequenced directly with methods well establishedin the art. Any primer pair derived from the combination of SEQ ID NO:11and SEQ ID NO:9 or the combination of SEQ ID NO:12 and SEQ ID NO:9 thatare useful in a DNA amplification method to produce an amplicondiagnostic for event MON 95379 or progeny thereof is an aspect of theinvention. Any single isolated DNA polynucleotide primer moleculecomprising at least 15 contiguous nucleotides of SEQ ID NO:11, or itscomplement that is useful in a DNA amplification method to produce anamplicon diagnostic for event MON 95379 or progeny thereof is an aspectof the invention. Any single isolated DNA polynucleotide primer moleculecomprising at least 15 contiguous nucleotides of SEQ ID NO:12, or itscomplement that is useful in a DNA amplification method to produce anamplicon diagnostic for plants comprising event MON 95379 or progenythereof is an aspect of the invention. Any single isolated DNApolynucleotide primer molecule comprising at least 15 contiguousnucleotides of SEQ ID NO:9, or its complement that is useful in a DNAamplification method to produce an amplicon diagnostic for event MON95379 or progeny thereof is an aspect of the invention.

Polynucleic acid amplification can be accomplished by any of the variouspolynucleic acid amplification methods known in the art, including thepolymerase chain reaction (PCR). Amplification methods are known in theart and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed.Innis et al., Academic Press. San Diego, 1990. PCR amplification methodshave been developed to amplify up to 22 kb (kilobase) of genomic DNA andup to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci.USA 91:5695-5699, 1994). These methods as well as other methods known inthe art of DNA amplification may be used in the practice of the presentinvention. The sequence of the heterologous DNA insert or flankinggenomic DNA sequence from corn event MON 95379 can be verified (andcorrected if necessary) by amplifying such DNA molecules from corn seedcontaining event MON 95379 DNA or corn plants grown from the corn seedcontaining event MON 95379 DNA deposited with the ATCC having accessionNo. PTA-125027, using primers derived from the sequences providedherein, followed by standard DNA sequencing of the PCR amplicon orcloned DNA fragments thereof.

The diagnostic amplicon produced by these methods may be detected by aplurality of techniques. One such method is Genetic Bit Analysis(Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNAoligonucleotide is designed that overlaps both the adjacent flankinggenomic DNA sequence and the inserted DNA sequence. The oligonucleotideis immobilized in wells of a microtiter plate. Following PCR of theregion of interest (using one primer in the inserted sequence and one inthe adjacent flanking genomic sequence), a single-stranded PCR productcan be hybridized to the immobilized oligonucleotide and serve as atemplate for a single base extension reaction using a DNA polymerase andlabeled dideoxynucleotide triphosphates (ddNTPs) specific for theexpected next base. Readout may be fluorescent or ELISA-based. A signalindicates presence of the transgene/genomic sequence due to successfulamplification, hybridization, and single base extension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method, anoligonucleotide is designed that overlaps the adjacent genomic DNA andinsert DNA junction. The oligonucleotide is hybridized tosingle-stranded PCR product from the region of interest (one primer inthe inserted sequence and one in the flanking genomic sequence) andincubated in the presence of a DNA polymerase. ATP, sulfurylase,luciferase, apyrase, adenosine 5′ phosphosulfate and luciferin. DNTPsare added individually and the incorporation results in a light signalthat is measured. A light signal indicates the presence of thetransgene/genomic sequence due to successful amplification,hybridization, and single or multi-base extension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon ofthe present invention. Using this method an oligonucleotide is designedthat overlaps the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence) and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. Single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene/genomic sequence due tosuccessful amplification, hybridization, and single base extension.

Real-time Polymerase Chain Reaction (PCR) is the ability to monitor theprogress of the PCR as it occurs (i.e., in real time). Data is collectedthroughout the PCR process, rather than at the end of the PCR. Inreal-time PCR, reactions are characterized by the point in time duringcycling when amplification of a target is first detected rather than theamount of target accumulated after a fixed number of cycles. In areal-time PCR assay, a positive reaction is detected by accumulation ofa fluorescent signal. The higher the starting copy number of the nucleicacid target, the sooner a significant increase in fluorescence isobserved. The cycle threshold (Ct value) is defined as the number ofcycles required for the fluorescent signal to cross the threshold (i.e.,exceeds background level). Ct levels are inversely proportional to theamount of target nucleic acid in the sample (i.e., the lower the Ctvalue, the greater the amount of target nucleic acid in the sample).

Taqman® (PE Applied Biosystems, Foster City, CA) is described as amethod of detecting and quantifying the presence of a DNA sequence usingreal-time PCR and is fully understood in the instructions provided bythe manufacturer. Briefly, a FRET oligonucleotide probe is designed thatoverlaps the genomic flanking and insert DNA junction. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking genomic sequence) are cycled in the presence of a thermalstablepolymerase and dNTPs. Hybridization of the FRET probe results incleavage and release of the fluorescent moiety away from the quenchingmoiety on the FRET probe. A fluorescent signal indicates the presence ofthe transgene/genomic sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996). Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermalstablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization.

DNA detection kits that are based on DNA amplification methods containDNA primer molecules that hybridize specifically to a target DNA andamplify a diagnostic amplicon under the appropriate reaction conditions.The kit may provide an agarose gel based detection method or any numberof methods of detecting the diagnostic amplicon that are known in theart. DNA detection kits can be developed using the compositionsdisclosed herein and are useful for identification of corn event MON95379 DNA in a sample and can be applied to methods for breeding cornplants containing event MON 95379 DNA. A kit that contains DNA primersthat are homologous or complementary to any portion of the corn genomicregion as set forth in SEQ ID NO:10 and to any portion of the insertedtransgenic DNA as set forth in SEQ ID NO:10 is an object of theinvention. The DNA molecules can be used in DNA amplification methods(PCR) or as probes in polynucleic acid hybridization methods, i.e.,southern analysis, northern analysis. Kits of the invention mayoptionally also comprise reagents or instructions for performing thedetection or diagnostic reactions described herein.

Probes and primers according to the invention may have complete sequenceidentity with the target sequence, although primers and probes differingfrom the target sequence that retain the ability to hybridizepreferentially to target sequences may be designed by conventionalmethods. In order for a nucleic acid molecule to serve as a primer orprobe it need only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed. Any conventional nucleic acidhybridization or amplification method can be used to identify thepresence of transgenic DNA from corn event MON 95379 in a sample. Probesand primers are generally at least about 11 nucleotides, at least about18 nucleotides, at least about 24 nucleotides, or at least about 30nucleotides or more in length. Such probes and primers hybridizespecifically to a target DNA sequence under stringent hybridizationconditions. Conventional stringency conditions are described by Sambrooket al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, DC (1985).

Any number of methods well known to those skilled in the art can be usedto isolate and manipulate a DNA molecule, or fragment thereof, disclosedin the invention, including thermal amplification methods. DNAmolecules, or fragments thereof, can also be obtained by othertechniques such as by directly synthesizing the fragment by chemicalmeans, as is commonly practiced by using an automated oligonucleotidesynthesizer.

The DNA molecules and corresponding nucleotide sequences provided hereinare therefore useful for, among other things, identifying corn event MON95379, detecting the presence of DNA derived from the transgenic cornevent MON 95379 in a sample, and monitoring samples for the presenceand/or absence of corn event MON 95379 or plant parts derived from cornplants comprising event MON 95379.

The invention provides corn plants, corn plant cells, corn seeds, cornplant parts (such as pollen, ovule, silk, spike, anther, cob, roottissue, stalk tissue, leaf tissue), corn progeny plants, and corncommodity products. These corn plants, corn plant cells, corn seeds,corn plant parts, corn progeny plants, and corn commodity productscontain a detectable amount of a polynucleotide of the invention, e.g.,such as a polynucleotide having at least one of the sequences providedas SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Cornplants, plant cells, seeds, plant parts, and progeny plants of theinvention may also contain one or more additional transgenes. Suchadditional transgene may be any nucleotide sequence encoding a proteinor RNA molecule conferring a desirable trait including but not limitedto increased insect resistance, increased water use efficiency,increased yield performance, increased drought resistance, increasedseed quality, and/or increased herbicide tolerance.

The invention provides corn plants, corn plant cells, corn seeds, cornplant parts (such as pollen, ovule, silk, spike, anther, cob, roottissue, stalk tissue, leaf tissue), corn progeny plants derived from atransgenic corn plant containing event MON 95379 DNA. A representativesample of corn seed containing event MON 95379 DNA has been depositedaccording to the Budapest Treaty with the American Type CultureCollection (ATCC®). The ATCC repository has assigned the Patent DepositDesignation PTA-125027 to the seed containing event MON 95379 DNA.

The invention provides a microorganism comprising a DNA molecule havingat least one sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, and SEQ ID NO:10 present in its genome. An example of sucha microorganism is a transgenic plant cell. Microorganisms, such as aplant cell of the invention, are useful in many industrial applications,including but not limited to: (i) use as research tool for scientificinquiry or industrial research; (ii) use in culture for producingendogenous or recombinant carbohydrate, lipid, nucleic acid, or proteinproducts or small molecules that may be used for subsequent scientificresearch or as industrial products; and (iii) use with modern planttissue culture techniques to produce transgenic plants or plant tissuecultures that may then be used for agricultural research or production.The production and use of microorganisms such as transgenic plant cellsutilizes modern microbiological techniques and human intervention toproduce a man-made, unique microorganism. In this process, recombinantDNA is inserted into a plant cell's genome to create a transgenic plantcell that is separate and unique from naturally occurring plant cells.This transgenic plant cell can then be cultured much like bacteria andyeast cells using modern microbiology techniques and may exist in anundifferentiated, unicellular state. The transgenic plant cell's newgenetic composition and phenotype is a technical effect created by theintegration of the heterologous DNA into the genome of the cell. Anotheraspect of the invention is a method of using a microorganism of theinvention. Methods of using microorganisms of the invention, such astransgenic plant cells, include (i) methods of producing transgeniccells by integrating recombinant DNA into the genome of the cell andthen using this cell to derive additional cells possessing the sameheterologous DNA; (ii) methods of culturing cells that containrecombinant DNA using modern microbiology techniques; (iii) methods ofproducing and purifying endogenous or recombinant carbohydrate, lipid,nucleic acid, or protein products from cultured cells; and (iv) methodsof using modern plant tissue culture techniques with transgenic plantcells to produce transgenic plants or transgenic plant tissue cultures.

Plants of the invention may pass along the event MON 95379 DNA,including transgene inserted in corn event MON 95379, to progeny. Asused herein, “progeny” includes any plant, plant cell, seed, and/orregenerable plant part containing the event MON 95379 DNA derived froman ancestor plant and/or comprising a DNA molecule having at least onesequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,and SEQ ID NO:10. Plants, progeny, and seeds may be homozygous orheterozygous for the transgene of event MON 95379. Progeny may be grownfrom seeds produced by a corn event MON 95379 containing plant and/orfrom seeds produced by a plant fertilized with pollen from a corn eventMON 95379 containing plant.

Progeny plants may be self-pollinated (also known as “selfing”) togenerate a true breeding line of plants, i.e., plants homozygous for thetransgene. Selfing of appropriate progeny can produce plants that arehomozygous for both added exogenous genes.

Alternatively, progeny plants may be out-crossed, e.g., bred withanother unrelated plant, to produce a varietal or a hybrid seed orplant. The other unrelated plant may be transgenic or non-transgenic. Avarietal or hybrid seed or plant of the invention may thus be derived bysexually crossing a first parent that lacks the specific and unique DNAof the corn event MON 95379 with a second parent comprising corn eventMON 95379, resulting in a hybrid comprising the specific and unique DNAof the corn event MON 95379. Each parent can be a hybrid or aninbred/varietal, so long as the cross or breeding results in a plant orseed of the invention, i.e., a seed having at least one allelecontaining the DNA of corn event MON 95379 and/or a DNA molecule havingat least one sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, and SEQ ID NO:10. Two different transgenic plants may thusbe crossed to produce hybrid offspring that contain two independentlysegregating, added, exogenous genes. For example, event MON 95379containing Cry1B.868 and Cry1Da_7 conferring insect resistance to corncan be crossed with other transgenic corn plants to produce a planthaving the characteristics of both transgenic parents. One example ofthis would be a cross of event MON 95379 containing Cry1B.868 and Cry1Da_7 conferring Lepidopteran resistance to corn with a plant having oneor more additional traits such as herbicide tolerance, insectresistance, or drought tolerance, resulting in a progeny plant or seedthat has resistance to Lepidopteran insect pests and has at least one ormore additional traits. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated, as isvegetative propagation. Descriptions of other breeding methods that arecommonly used for different traits and crops can be found in one ofseveral references, e.g., Fehr, in Breeding Methods for CultivarDevelopment, Wilcox J. ed., American Society of Agronomy, Madison WI(1987).

Plants, progeny, seeds, cells and plant parts of the invention may alsocontain one or more additional corn trait(s) or transgenic events,particularly those introduced by crossing a corn plant containing cornevent MON 95379 with another corn plant containing the additionaltrait(s) or transgenic events. Such trait(s) or transgenic eventsinclude, but are not limited to, increased insect resistance, herbicidetolerance, increased water use efficiency, increased yield performance,increased drought resistance, increased seed quality, improvednutritional quality, hybrid seed production, or disease or fungalresistance. Corn transgenic events are known to those of skill in theart. For example, a list of such traits is provided by the United StatesDepartment of Agriculture's (USDA) Animal and Plant Health InspectionService (APHIS) and can be found on their website: www.aphis.usda.gov.Two or more transgenic events may thus be combined in a progeny seed orplant by crossing two parent plants each comprising one or moretransgenic events, collecting the progeny seed, and selecting forprogeny seed or plants that contain the two or more transgenic events.These steps may be repeated until the desired combination of transgenicevents in a progeny is achieved. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated, and isvegetative propagation.

The invention provides a plant part that is derived from corn plantscomprising event MON 95379. As used herein, a “plant part” refers to anypart of a plant which is comprised of material derived from a corn plantcomprising event MON 95379. Plant parts include but are not limited topollen, ovule, silk, spike, anther, cob, root tissue, stalk tissue, andleaf tissue. Plant parts may be viable, nonviable, regenerable, and/ornonregenerable.

The invention provides a commodity product that is derived from cornplants comprising event MON 95379 and that contains a detectable amountof a nucleic acid specific for event MON 95379. As used herein, a“commodity product” refers to any composition or product which iscomprised of material derived from a corn plant, whole or processed cornseed, or one or more plant cells and/or plant parts containing the cornevent MON 95379 DNA. Nonviable commodity products include but are notlimited to nonviable seeds, whole or processed seeds, seed parts, andplant parts; animal feed comprising corn, corn oil, corn meal, cornflour, corn flakes, corn bran, pasta made with corn, corn biomass, andfuel products produced using corn and corn parts. Viable commodityproducts include but are not limited to seeds, plants, and plant cells.The corn plants comprising event MON 95379 can thus be used tomanufacture any commodity product typically acquired from corn. Any suchcommodity product that is derived from corn plants comprising event MON95379 may contain at least a detectable amount of the specific andunique DNA corresponding to corn event MON 95379, and specifically maycontain a detectable amount of a polynucleotide comprising a DNAmolecule having at least one sequence selected from SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Any standard method ofdetection for nucleotide molecules may be used, including methods ofdetection disclosed herein. A commodity product is with the scope of theinvention if there is any detectable amount of a DNA molecule having atleast one sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, and SEQ ID NO:10 in the commodity product.

The corn plants, corn plant cells, corn seeds, corn plant parts (such aspollen, ovule, silk, spike, anther, cob, root tissue, stalk tissue, leaftissue), corn progeny plants, and commodity products of the inventionare therefore, useful for, among other things, growing plants for thepurpose of producing seed and/or plant parts comprising corn event MON95379 for agricultural purposes, producing progeny comprising corn eventMON 95379 for plant breeding and research purposes, use withmicrobiological techniques for industrial and research applications, andsale to consumers.

Methods for producing an insect resistant corn plant comprising the DNAsequences specific and unique to event MON 95379 of the invention areprovided. Transgenic plants used in these methods may be homozygous orheterozygous for the transgene. Progeny plants produced by these methodsmay be varietal or hybrid plants; may be grown from seeds produced bycorn event MON 95379 containing plant and/or from seeds produced by aplant fertilized with pollen from a corn event MON 95379 containingplant; and may be homozygous or heterozygous for the transgene. Progenyplants may be subsequently self-pollinated to generate a true breedingline of plants, i.e., plants homozygous for the transgene, oralternatively may be out-crossed, e.g., bred with another unrelatedplant, to produce a varietal or a hybrid seed or plant.

Methods of detecting the presence of DNA derived from a corn cell, corntissue, corn seed, or corn plant comprising corn event MON 95379 in asample are provided. One method consists of (i) extracting a DNA samplefrom at least one corn cell, corn tissue, corn seed, or corn plant; (ii)contacting the DNA sample with at least one primer that is capable ofproducing DNA sequence specific to event MON 95379 DNA under conditionsappropriate for DNA sequencing; (iii) performing a DNA sequencingreaction; and then (iv) confirming that the nucleotide sequencecomprises a nucleotide sequence specific for event MON 95379, of theconstruct comprised therein, such as one selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ IDNO:10. Another method consists of (i) extracting a DNA sample from atleast one corn cell, corn tissue, corn seed, or corn plant; (ii)contacting the DNA sample with a primer pair that is capable ofproducing an amplicon from event MON 95379 DNA under conditionsappropriate for DNA amplification; (iii) performing a DNA amplificationreaction; and then (iv) detecting the amplicon molecule and/orconfirming that the nucleotide sequence of the amplicon comprises anucleotide sequence specific for event MON 95379, such as one selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, and SEQ ID NO:10. The amplicon should be one that is specific forevent MON 95379, such as an amplicon that comprises SEQ ID NO:1, or SEQID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:6,or SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:9, or SEQ ID NO:10. Thedetection of a nucleotide sequence specific for event MON 95379 in theamplicon is determinative and/or diagnostic for the presence of the cornevent MON 95379 specific DNA in the sample. An example of a primer pairthat is capable of producing an amplicon from event MON 95379 DNA underconditions appropriate for DNA amplification is provided as SEQ ID NO:15and SEQ ID NO:16. Other primer pairs may be readily designed by one ofskill in the art and would produce an amplicon comprising SEQ ID NO:1,or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, or SEQID NO:6, or SEQ ID NO:7, or SEQ ID NO:8 wherein such a primer paircomprises at least one primer within the genomic region flanking theinsert and a second primer within the insert. Another method ofdetecting the presence of DNA derived from a corn cell, corn tissue,corn seed, or corn plant comprising corn event MON 95379 in a sampleconsists of (i) extracting a DNA sample from at least one corn cell,corn tissue, corn seed, or corn plant; (ii) contacting the DNA samplewith a DNA probe specific for event MON 95379 DNA; (iii) allowing theprobe and the DNA sample to hybridize under stringent hybridizationconditions; and then (iv) detecting hybridization between the probe andthe target DNA sample. An example of the sequence of a DNA probe that isspecific for event MON 95379 is provided as SEQ ID NO:17. Other probesmay be readily designed by one of skill in the art and would comprise atleast one fragment of genomic DNA flanking the insert and at least onefragment of insert DNA such as the sequence provided in SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, and SEQ ID NO:10. Detection of probe hybridization tothe DNA sample is diagnostic for the presence of corn event MON 95379specific DNA in the sample. Absence of hybridization is alternativelydiagnostic of the absence of corn event MON 95379 specific DNA in thesample.

DNA detection kits are provided that are useful for the identificationof corn event MON 95379 DNA in a sample and can also be applied tomethods for breeding corn plants containing the appropriate event DNA.Such kits contain DNA primers and/or probes comprising fragments of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Oneexample of such a kit comprises at least one DNA molecule of sufficientlength of continuous nucleotides of SEQ ID NO:10 to function as a DNAprobe useful for detecting the presence and/or absence of DNA derivedfrom transgenic corn plants comprising event MON 95379 in a sample. TheDNA derived from transgenic corn plants comprising event MON 95379 wouldcomprise a DNA molecule having at least one sequence selected from SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. A DNAmolecule sufficient for use as a DNA probe is provided that is usefulfor determining, detecting, or diagnosing the presence and/or absence ofcorn event MON 95379 DNA in a sample is provided as SEQ ID NO: 17. Otherprobes may be readily designed by one of skill in the art and shouldcomprise at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25, at least 26, at least 27, at least 28, at least 29, at least30, at least 31, at least 32, at least 33, at least 34, at least 35, atleast 36, at least 37, at least 38, at least 39, or at least 40contiguous nucleotides of SEQ ID NO:10 and be sufficiently unique tocorn event MON 95379 DNA in order to identify DNA derived from theevent.

Another type of kit comprises a primer pair useful for producing anamplicon useful for detecting the presence and/or absence of DNA derivedfrom transgenic corn event MON 95379 in a sample. Such a kit wouldemploy a method comprising contacting a target DNA sample with a primerpair as described herein, then performing a nucleic acid amplificationreaction sufficient to produce an amplicon comprising a DNA moleculehaving at least one sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:10 and then detecting the presenceand/or absence of the amplicon. Such a method may also includesequencing the amplicon or a fragment thereof, which would bedeterminative of, i.e., diagnostic for, the presence of the corn eventMON 95379 specific DNA in the target DNA sample. Other primer pairs maybe readily designed by one of skill in the art and should comprise atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, or at least 30contiguous nucleotides of sequences provided in, but not limited to SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, and besufficiently unique to corn event MON 95379 DNA in order to identify DNAderived from the event.

The kits and detection methods of the invention are useful for, amongother things, identifying corn event MON 95379, selecting plantvarieties or hybrids comprising corn event MON 95379, detecting thepresence of DNA derived from the transgenic corn plant comprising eventMON 95379 in a sample, and monitoring samples for the presence and/orabsence of corn plants comprising event MON 95379, or plant partsderived from corn plants comprising event MON 95379.

The sequences of the heterologous DNA insert, junction sequences, orflanking sequence from corn event MON 95379 can be verified (andcorrected if necessary) by amplifying such sequences from the eventusing primers derived from the sequences provided herein followed bystandard DNA sequencing of the amplicon or of the cloned DNA.

Methods of detecting the zygosity of the transgene allele of DNA derivedfrom a corn cell, corn tissue, corn seed, or corn plant comprising cornevent MON 95379 in a sample are provided. One method consists of (i)extracting a DNA sample from at least one corn cell, corn tissue, cornseed, or corn plant; (ii) contacting the DNA sample with a primer pairthat is capable of producing a first amplicon diagnostic for event MON95379; (iii) contacting the DNA sample with a primer pair that iscapable of producing a second amplicon diagnostic for native corngenomic DNA not comprising event MON 95379; (iv) performing a DNAamplification reaction; and then (v) detecting the amplicons, whereinthe presence of only the first amplicon is diagnostic of a homozygousevent MON 95379 DNA in the sample, and the presence of both the firstamplicon and the second amplicon is diagnostic of a corn plantheterozygous for event MON 95379 allele. An exemplary set of primerspairs are presented as SEQ ID NO:15 and SEQ ID NO:16 which produce anamplicon diagnostic for event MON 95379; and SEQ ID NO:15 and SEQ IDNO:27 which produces an amplicon diagnostic for non-inserted wild-typecorn genomic DNA not comprising event MON 95379. A set of probes canalso be incorporated into such an amplification method to be used in areal-time PCR format using the primer pair sets described above. Anexemplary set of probes are presented as SEQ ID NO:17 (diagnostic forthe amplicon for the event MON 95379) and SEQ ID NO:28 (diagnostic forthe amplicon for wild-type corn genomic DNA not comprising event MON95379).

Another method for determining zygosity consists of (i) extracting a DNAsample from at least one corn cell, corn tissue, corn seed, or cornplant; (ii) contacting the DNA sample with a probe set which contains atleast a first probe that specifically hybridizes to event MON 95379 DNAand at least a second probe that specifically hybridizes to corn genomicDNA that was disrupted by insertion of the heterologous DNA of event MON95379 and does not hybridize to event MON 95379 DNA; (iii) hybridizingthe probe set with the sample under stringent hybridization conditions,wherein detecting hybridization of only the first probe under thehybridization conditions is diagnostic for a homozygous allele of eventMON 95379 DNA in the sample; and wherein detecting hybridization of boththe first probe and the second probe under the hybridization conditionsis diagnostic for a heterozygous allele of event MON 95379 in a DNAsample.

Yet another method for determining zygosity consists of (i) extracting aDNA sample from at least one corn cell, corn tissue, corn seed, or cornplant; (ii) contacting the DNA sample with a primer pair that is capableof producing an amplicon of one of the toxin coding sequences encodingCry1B.868 or Cry1Da_7; (iii) contacting the DNA sample with a primerpair that is capable of producing an amplicon of an internal standardknown to be single-copy and homozygous in the corn plant; (iv)contacting the DNA sample with a probe set which contains at least afirst probe that specifically hybridizes to one of the toxin codingsequences encoding Cry1B.868 or Cry1Da_7, and at least a second probethat specifically hybridizes to the internal standard genomic DNA knownto be single-copy and homozygous in the corn plant; (v) performing a DNAamplification reaction using real-time PCR and determining the cyclethresholds (Ct values) of the amplicon corresponding to the toxin codingsequence and the single-copy, homozygous internal standard; (vi)calculating the difference (ΔCt) between the Ct value of thesingle-copy, homozygous internal standard amplicon and the Ct value ofthe toxin coding sequence amplicon; and (vii) determining zygosity,wherein a ΔCt of around zero (0) indicates homozygosity of the insertedT-DNA and a ΔCt of around one (1) indicates heterozygosity of theinserted T-DNA. Heterozygous and homozygous events are differentiated bya ΔCt value unit of approximately one (1). Given the normal variabilityobserved in real-time PCR due to multiple factors such as amplificationefficiency and ideal annealing temperatures, the range of “about one(1)” is defined as a ΔCt of 0.75 to 1.25. Primer pairs and probes forthe above method for determining zygosity can either amplify and detectamplicons from the Cry1B.868 coding sequence and internal standard, oramplify and detect amplicons from the Cry1Da_7 coding sequence andinternal standard. Exemplary primer pairs for the detection of theamplicons corresponding to the Cry1B.868 coding sequence and internalstandard are presented as SEQ ID NO:18 combined with SEQ ID NO:19(internal standard) and SEQ ID NO:21 combined with SEQ ID NO:22(Cry1B.868). The accompanying exemplary probes are presented as SEQ IDNO:20 (internal standard) and SEQ ID NO:23 (Cry1B.868). Exemplary primerpairs for the detection of the amplicons corresponding to the Cry1Da_7coding sequence and internal standard are presented as SEQ ID NO:18combined with SEQ ID NO:19 (internal standard) and SEQ ID NO:24 combinedwith SEQ ID NO:25 (Cry1Da_7). The accompanying exemplary probes arepresented as SEQ ID NO:20 (internal standard) and SEQ ID NO:26(Cry1Da_7).

DEPOSIT INFORMATION

A deposit of a representative sample of corn seed containing event MON95379 was made on Apr. 20, 2018 according to the Budapest Treaty withthe American Type Culture Collection (ATCC) having an address at 10801University Boulevard, Manassas, Virginia USA, Zip Code 20110, andassigned ATCC Accession No. PTA-125027. Access to the deposits will beavailable during the pendency of the application to the Commissioner ofPatents and Trademarks and persons determined by the Commissioner to beentitled thereto upon request. Upon issuance of the patent, allrestrictions upon availability to the public will be irrevocablyremoved. The deposit has been accepted under the Budapest Treaty andwill be maintained in the depository for a period of thirty (30) years,or five (5) years after the last request, or for the effective life ofthe patent, whichever is longer, and will be replaced as necessaryduring that period.

EXAMPLES

The following Examples are included to more fully describe theinvention. Summarized are the construction and testing of one hundredand twenty-five (125) constructs, the production of about ten thousandseven hundred and eighty-five (10,785) events (both proof of concept andcommercial), and the analysis of hundreds of thousands of individualplants over six (6) years through the rigorous molecular, agronomic, andfield testing required for the creation and selection of corn event MON95379.

The Examples demonstrate certain preferred embodiments of the invention.It should be appreciated by those of skill in the art that thetechniques disclosed in the Examples that follow represent approachesthe inventors have found function well in the practice of the invention,and thus can be considered to constitute examples of preferred modes forits practice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Expression Cassette Testing, Construct Design, Plant Testingand Construct Selection

Transgene expression in plants is influenced by numerous differentfactors. The right combination of insecticidal proteins and differentexpression elements driving expression in plants, while not resulting inoff-phenotypes, must be found. Further, beyond the expression elementsthemselves and their combination and orientation in a cassette, theexpression of transgenes in plants is known to be influenced bychromosomal insertion position, perhaps due to chromatin structure(e.g., heterochromatin) or the proximity of transcriptional regulationelements (e.g., enhancers) close to the integration site (Kurt Weisinget al., (1988) Foreign genes in plants: transfer, structure, expressionand applications. Annu. Rev. Genet. 22: 421-77). For example, it hasbeen observed in plants and in other organisms that there may be widevariation in the levels of expression of an introduced gene from thesame construct among events with different chromosomal insertionpositions. Different chromosomal insertion positions may also producedifferences in spatial or temporal patterns of expression, that may notcorrespond to the patterns expected from transcriptional regulatoryelements present in the introduced gene construct.

For these reasons, it is often necessary to create and screen a largenumber of constructs and transformation events in order to identify aconstruct, and then an event, which demonstrates optimal expression ofthe introduced genes of interest, while also not producing agronomic orphenotypic off-types.

For these reasons, the development of a transgenic corn plant comprisinginsecticidal proteins that were active against Lepidopterans without anynegative effects on agronomics, yield, or stacking viability requiredextensive research, development, and analysis. Specifically, over a six(6) year period, approximately ten thousand, seven hundred eight-five(10,785) proof of concept and commercial transgenic events derived fromone hundred twenty-five (125) different plasmid vector constructs weredeveloped, tested, and analyzed.

This Example describes the design and testing in corn plants of onehundred and twenty five (125) different constructs to identify thepreferred construct for event creation. Each construct varied withrespect to the coding sequences for the insecticidal proteins and thetranscriptional regulatory elements. Testing was done to select the bestconstruct for use in expressing the insecticidal proteins in plants.Each construct had a unique configuration, varying by expressioncassette composition (both insecticidal proteins and expressionelements), orientation, and whether or not proteins were targeted to thechloroplast.

In an initial proof of concept and developmental stage, one hundredseventeen (117) constructs comprising different combinations oftwenty-six (26) distinct promoters, twenty-six (26) distinct introns,and ten (10) distinct insect toxin coding sequences, were used togenerate approximately six thousand (6,000) transformed events. Afterinitial molecular characterization for the presence of the transgene(s),five thousand fifty-two (5,052) single and double-copy transformed cornevents were selected for further characterization and efficacy testing.These events were evaluated for phenotypic or agronomic off-types, thelevel of expression of the insect toxin proteins, and efficacy againstselected Lepidopteran insect pest species. The resulting efficacy andprotein expression data, along with any information regarding phenotypicand agronomic off-types was used to eliminate inefficacious proteins,expression elements and combinations, and was used to design a smallernumber of binary commercial transformation plasmid constructs to be usedin the next phase of development.

In the next phase of development, eight (8) new constructs were created.These constructs comprised combinations of two (2) to four (4) insecttoxin transgene expression cassettes in different orientations(convergent or divergent). These eight (8) constructs were used togenerate a total of five thousand seven hundred thirty-three (5,733)transformed events (also referred to as “transformants”). After shootformation in culture, a subset of the transformed events were selectedbased upon visual characteristics and early molecular analysis. A totalof eight hundred twenty-three (823) transformed events were selected andtransplanted to pots and grown for further study.

The resulting R₀ generation transformed events were analyzed forefficacy against selected Lepidopteran species, toxin proteinexpression, plant health, seed return, and phenotypic and agronomicoff-types. The R₀ generation events were also characterized molecularlyto ensure cassette intactness and proper insertion in the corn genome.Many of the events were dropped from testing due to failure to pass theagronomic analysis and molecular characterization testing. In addition,one (1) of the eight (8) constructs was dropped from further study atthis R₀ stage because it produced events with off-phenotypes. Inaddition to these agronomic problems, later mode of action (“MOA”)studies conducted demonstrated that an insect toxin protein contained inthis construct demonstrated an overlapping MOA to acommercially-available protein.

Mode of Action studies were conducted on one of the insect proteinscommon to four (4) of the eight (8) constructs. These studiesdemonstrated that this insect protein had an overlapping MOA to acommercially-available protein. Proteins that demonstrate an analogousor overlapping MOA to a currently utilized commercial insecticidalprotein are not desirable because of resistance development, which couldrender a protein with a similar MOA ineffective against insectpopulations. As such, these four (4) constructs, and the events arisingtherefrom, were dropped. As noted previously, one (1) of the four (4)dropped constructs also produced events with off-phenotypes at the R₀stage.

In the next stage of development, one hundred fifty (150) events derivedfrom the remaining four (4) constructs were further evaluated at the F₁(heterozygous hybrid)/R₁ (homozygous inbred) and R₂ generation forefficacy, seed return and segregation, phenotypic and agronomicoff-types, and further molecular characterization. Two (2) constructsfrom the remaining four (4) constructs were dropped from further studyin this stage for failure to meet one or more of the criteria foradvancement, leaving events derived from (2) constructs for furtherevaluation.

Seventy-seven (77) events, forty-one (41) derived from the constructused to generate event MON 95379 (“Construct MON95”) and thirty-six (36)events derived from the other construct (“Construct 1”), were evaluatedas R₂ inbreds and F₁ hybrids for efficacy, seed return and segregation,phenotypic and agronomic off-types and further molecularcharacterization. Based upon the results of these evaluations, eventsassociated with Construct 1 were de-prioritized, shelved and stored.

Thus, numerous rounds of testing and comparison of various constructsrevealed that the transgene cassette provided as SEQ ID NO:13, ConstructMON95, was the best option for efficacy against the Lepidopteran pestspecies Fall Armyworm (FAW, Spodoptera frugiperda), Corn Earworm (CEW,Helicoverpa zea), Southwestern Corn Borer (SWCB, Diatraea grandiosella),Surgarcane Borer (SCB, Diatraea saccharalis), and Lesser Cornstalk Borer(LSCB, Elasmopalpus lignosellus), with the best molecularcharacterization and agronomic performance.

Table 2 illustrates the number of transformed events derived(“Plugged”), the number of transformed events selected for growth as R0events (“Transplanted”), and the points at which each respectiveconstruct was dropped in the evaluation, research and developmentprocess that led to the selection of Construct MON95.

TABLE 2 Event construct selection. Construct Plugged TransplantedDropped Construct MON 95 1202 210 Construct 1 799 173 Shelved based onR₂ and F₁ data Construct 2 679 113 R₂/F1/R₁ Construct 3 1344 20 MOA/R₀Construct 4 232 41 R₂/F₁/R₁ Construct 5 544 88 MOA Construct 6 584 95MOA Construct 7 349 83 MOA

Example 2 Field Trials, Molecular Testing and Event Selection

This Example describes the molecular characterization, analysis, andtesting in field trials of events created with Construct MON95 inmultiple locations over several years, which lead to the selection ofthe final event, MON 95379.

Table 3 illustrates the process used to select the final event, MON95379. At the commercial transformation R₀ screen, two hundred ten (210)R₀ transformed events from Construct MON95 were derived and selected forgrowth. Of the initial two hundred ten (210) selected R₀ transformedevents, one hundred forty-seven events (147) were dropped due toconcerns regarding efficacy, protein expression, seed return and planthealth, or molecular characterization. This left sixty-three (63) eventsfor assay and testing in the next stage of development, the F₁ Screenand the R₁ Nursery stage. In this stage, eleven (11) events were droppeddue to efficacy concerns in the greenhouse testing. Another three (3)events were dropped because of insufficient return of seed from thenursery and/or segregation analysis of the resulting seed. Finally,another five (5) events were removed due to issues discovered inmolecular characterization and three (3) events were removed due toissues discovered in molecular southern analysis, leaving forty-one (41)events for assay in the next generation. At the R₂/F₁ stage of testing,two (2) of the remaining forty-one (41) events were dropped due toissues discovered in further molecular southern characterization,leaving thirty-nine (39) events.

The remaining thirty-nine (39) events were advanced in two differentconcurrent parallel testing stages: 1) further field trials' and 2)Cre-excision of the selection cassette and the production of goldstandard seed. Events were dropped in each of these concurrent paralleltesting stages.

During Cre-excision, eleven (11) events were dropped due to issuesdiscovered in molecular characterization after cre-excision of theglyphosate selection cassette. Further, another six (6) events weredropped due to issues discovered in molecular characterization duringgold standard seed production.

During the concurrent field testing, based on data collected from the2016 U.S. Field Trails, another four (4) events were dropped due toefficacy concerns and another twelve (12) events were dropped due toagronomic concerns. Then, based on data collected from the Brazil FieldTrials, another event was dropped due to efficacy concerns. Next,bioinformatic analysis conducted during the 2017 U.S. Field Trialsresulted in the removal of another three (3) events from furthertesting, leaving two events: Event 1 and MON 95379. After furtheranalysis of the agronomics of the events from multiple field trials inthe U.S., Brazil, Argentina and Puerto Rico, event MON 95379 wasselected as the event for commercialization because it ranked higherthan Event 1 when all the characteristics of molecular characterization,protein expression, efficacy and agronomics of each event were compared.

TABLE 3 MON 95379 event selection. Events Events Remaining Stage AssayRemoved 210 Comm. TFN R₀ Efficacy 21 63 Screen Expression 6 SeedReturn/plant health 51 Molecular characterization 69 R₁/F₁ GreenhouseEfficacy 11 41 Molecular characterization 5 Nursery return/segregation 3Molecular southern 3 R₂/F₁ Molecular southern 2 39 Cre ExcisionMolecular characterization after 11 28 CP4 excision Gold Standard SeedMolecular characterization 6 22 U.S. Field 2016 Efficacy 4 6 Agronomics12 Brazil Field Efficacy 1 5 U.S. Field 2017 Bioinformatic molecularanalysis 3 2 Commercial Selection Further analysis of molecular 1 MON95379 characterization, protein expression, efficacy and agronomics frommultiple field trials

Example 3 Cre-Excision of the Glyphosate Selection Cassette in CornEvent MON 95379

This Example describes the removal of the glyphosate selection cassettefrom corn event MON 95379 through in vivo Cre-excision. The glyphosateselection cassette was used to select transformed events. By removal ofthe selection cassette, a “marker-free” event was created wherein onlythe insecticidal protein expression cassettes remained in the finalevent.

FIG. 3 illustrates the breeding process used to generate the marker-freeevent MON 95379 corn event. Corn variety LH244 immature embryos weretransformed using an Agrobacterium-mediated transformation process withConstruct MON95 (presented as SEQ ID NO:13, and illustrated in FIG. 2 ).Construct MON95 comprises three (3) expression cassettes: two (2)expression cassettes for the expression of the insecticidal proteinsCry1B.868 and Cry1Da_7, and a single cassette used for the selection oftransformed plant cells using glyphosate selection. The selectioncassette was flanked on both sides with LoxP Cre-recombinase recognitionsites.

After transformation, the R₀ transformants were self-pollinated for two(2) generations, during which time many events were removed based uponvarious assays such as efficacy, protein expression, seed return andplant health, and molecular characterization. By the R₂ generation,thirty-nine (39) events remained from the initial two hundred ten (210)events. The thirty-nine (39) homozygous R₂ generation events were bredwith an elite line of transformed corn plants expressing Cre-recombinaseenzyme, derived from Enterobacteria phage P1.

This stage in which R₂ generation events were bred with plantsexpressing Cre-recombinase is identified as “Cre Cross”. Specifically inthis stage, de-tasseled (female) R₂ generation plants homozygous for SEQID NO:13 were cross-pollinated with transgenic corn plants (male)homozygous for a transgene cassette used for expression ofCre-recombinase enzyme. The Cre-recombinase expressing male donor pollengerminates after landing on the silk tissue of the female plantcomprising SEQ ID NO:13. Once the pollen tube enters the embryo sac, thepollen tube ruptures, setting free the two sperms of the Cre-recombinaseexpressing male donor. The nucleus of one sperm fuses with the eggnucleus, forming the zygote. The other sperm nucleus fuses with one ofthe two polar nuclei which in turn fuses with the other polar nucleus,thereby establishing the primary endosperm nucleus.

Thus, in using the Cre-recombinase expressing plant as the male pollendonor, both the embryo and endosperm of the resulting cross will expressCre-recombinase as the cells divide and develop and become a corn kernel(i.e., seed). The Cre-recombinase binds to inverted repeats in the LoxPsite and catalyzes a crossover in an eight-base pair spacer region ofthe two LoxP sites that flank the expression cassette, resulting in theexcision of the marker cassette with one LoxP site remaining in theintegrated T-DNA due to recombination (see FIG. 2 , “Inserted T-DNAAfter Cre-Excision”).

The F₁ progeny resulting from the Cre Cross were selected for theabsence of the CP4 selection cassette and allowed to self-pollinate.Through this process, the two alleles—the Cre-recombinase allele and theallele for the T-DNA used to generate event MON 95379—segregate in theresulting F₂ population, resulting in progeny homozygous or heterozygousfor one or both alleles.

The F₂ progeny which demonstrated the absence of the Cre-recombinaseallele and homozygosity for SEQ ID NO:9, the transgenic inserted T-DNAafter Cre-excision, were selected. These selected F₂ progeny wereself-pollinated, giving rise to an F₃ generation homozygous for SEQ IDNO:9.

A further self-pollination resulted in F₃ progeny seed (F₄ seed) whichwere assayed for purity, and were designated as “Gold Standard Seed.” F₄was the first generation of gold standard seed.

Excision of the glyphosate selection marker cassette did not affect theexpression of Cry1B.868 and Cry1Da_7. Removing the glyphosate selectioncassette from corn event MON 95379 through Cre-excision provided atransgenic corn event which is resistant to Lepidopteran pests withoutadding tolerance to glyphosate in the final event. This “marker-free”event assures flexibility when building corn breeding stacks with othercorn transgenic events to provide a multiplicity of productsincorporating event MON 95379 and allowing multiple options forproviding additional traits in the final breeding stacks.

Example 4 Corn Event MON 95379 Demonstrates Resistance to theLepidopteran Insect Pests Fall Armyworm, Corn Earworm, Southwestern CornBorer, Sugarcane Borer

This Example describes the activity of the MON 95379 event againstLepidopteran insect pests. The insect toxin proteins Cry1B.868 andCry1Da_7, when expressed together in corn event MON 95379, provideresistant to Fall Armyworm (Spodoptera frugiperda), Corn Earworm(Helicoverpa zea), Southwestern Corn Borer (Diatraea grandiosella), andSurgarcane Borer (Diatraea saccharalis).

After transformation and insertion of Construct MON95, forty-one (41) R₀events were selected for bioassay using leaf discs. Bioassays usingplant leaf disks were performed analogous to those described in U.S.Pat. No. 8,344,207. A non-transformed LH244 corn plant was used toobtain tissue to be used as a negative control. Plates comprising wellswith one insect per leaf disc in each well were incubated for three (3)days. After three (3) days, the plates were examined. If at least fiftypercent (50%) of the leaf disc in the negative controls was consumed,measurements were taken of the transgenic event leaf discs. If less thanfifty percent (50%) of the leaf discs in the negative controls had notyet been consumed, the insects were allowed to continue feeding untilthe fifty percent (50%) target was achieved. Measurements of leaf damage(“leaf damage ratings” or “LDR”) and mortality were taken for each well.An average of each measure was determined. The leaf damage ratingsranged from one (1) to eleven (11) and reflect a percentage of theconsumed leaf disc. Table 4 shows the leaf damage rating scale used forthe R₀ leaf disc assays. On this rating scale, the negative controlswill always have an LDR of at least 10.

TABLE 4 Leaf Damage Ratings (LDR) scale for R₀ leaf disc assays. Amountof Leaf Damage feeding Rating (LDR) damage 1  ≤5% 2 ≤10% 3 ≤15% 4 ≤20% 5≤25% 6 ≤30% 7 ≤35% 8 ≤40% 9 ≤45% 10 ≤50% 11 >50%

Table 5 shows the mean leaf damage ratings for the forty-one (41) eventstransformed with Construct MON95, including the MON 95379 event. As canbe seen in Table 5, expression of the two insecticidal proteins,Cry1B.868 and Cry1Da_7, provided resistance to Fall Armyworm (FAW), CornEarworm (CEW), and Southwestern Corn Borer (SWCB). The LDRs for thenegative controls were between 10 and 11. The FAW and SWCB consumed onlyapproximately five percent (5%) of the event MON 95379 leaf disc incomparison to the negative controls which consumed at least fiftypercent (50%) of the leaf disc. With respect to CEW, only approximately6.25% of the leaf discs were consumed in comparison to the negativecontrols which consumed at least fifty percent (50%) of the leaf disc.In addition, one hundred percent (100%) of the FAW and CEW were killedafter consuming the event MON 95379 containing leaf discs.

TABLE 5 Mean Leaf Damage Rating (LDR) scores and Mean Mortality for R₀plants expressing Cry1B.868 and Cry1Da_7. FAW CEW SWCB Mean Mean MeanEvent LDR LDR LDR MON 95379 1.00 1.25 1.00 Event 2 1.00 2.00 1.00 Event3 1.00 1.00 1.00 Event 4 1.00 1.25 1.00 Event 5 1.00 1.50 1.00 Event 61.00 3.25 1.00 Event 7 1.00 2.25 1.00 Event 8 1.00 1.50 1.00 Event 91.00 1.75 1.00 Event 10 1.00 2.25 1.25 Event 11 1.00 1.75 1.00 Event 121.00 1.25 1.00 Event 13 1.00 1.25 1.00 Event 14 1.00 3.75 1.00 Event 151.00 1.00 1.00 Event 16 1.00 1.25 1.25 Event 17 1.00 3.00 1.00 Event 181.00 1.25 1.00 Event 19 1.00 1.75 1.25 Event 20 1.00 1.00 1.25 Event 211.00 5.00 1.00 Event 22 1.00 1.33 1.00 Event 23 1.00 1.00 1.00 Event 241.00 1.50 1.00 Event 25 1.00 1.25 1.00 Event 26 1.00 2.33 1.25 Event 271.00 1.25 1.00 Event 28 1.00 1.50 1.00 Event 29 1.00 1.00 1.00 Event 301.00 2.00 1.00 Event 31 1.00 2.50 1.50 Event 32 1.00 1.00 1.00 Event 331.00 1.67 1.00 Event 34 1.00 3.00 1.00 Event 35 1.00 1.25 1.00 Event 361.00 1.50 1.00 Event 37 1.00 2.50 1.00 Event 38 1.00 3.50 1.25 Event 391.00 1.00 1.25 Event 40 1.00 1.00 1.00 Event 41 1.00 1.25 1.00

The forty-one (41) events were crossed with non-transgenic 93IDI3variety plants. F₁ heterozygous progeny plants were selected thatcomprised Construct MON95. Around five (5) F₁ plants for each event wereartificially infested in a greenhouse for each insect pest species. Withrespect to FAW, approximately forty (40) neonates were used to infesteach F₁ plant in the V6 to V8 stage whorl. With respect to SWCB,approximately thirty (30) neonates were used to infest the F₁ plant inthe V6 to V10 stage whorl. Measures of leaf damage for FAW and SWCB weretaken approximately fourteen (14) days after infestation. Tables 6 and 7show the damage rating scales used to assess the leaf damage.

TABLE 6 Leaf damage rating scale for corn plants infested with FAW. LeafDamage Rating (LDR) Description 0 No visible damage 1 Only pinholelesions present on whorl leaves 2 Pinholes and small, circular lesionspresent on whorl leaves 3 Small, circular lesions and 1-3 small,elongated lesions present on whorl and furl leaves 4 4-6 small tomid-sized, elongated lesions present on 1-3 whorl and furl leaves 5 4-6large, elongated lesions present on 1-3 whorl and furl leaves and/or 1-3small or mid-sized, uniform to irregular lesions, or both eaten fromwhorl and/or furl leaves 6 4-6 large, elongated lesions present on 4-6whorl and furl leaves and/or 4-6 large uniform to irregular shaped holeseaten from whorl and furl leaves 7 7+ elongated lesions of all sizespresent on several whorl and furl leaves plus 4-6 large uniform toirregular shaped holes eaten from the whorl and furl leaves 8 7+elongated lesions of all sizes on most whorl and furl leaves plus 7+mid- to large-sized uniform to irregular-shaped holes eaten from thewhorl and furl leaves 9 Whorl and furl leaves almost totally destroyedas well as the plant showing signs of stunting

TABLE 7 Leaf damage rating scale for corn plants infested with SWCB.Leaf Damage Rating (LDR) Description 0 No lesions 1 Small amount of pinor fine holes on leaves 2 Small amount of shot-hole injury on a fewpresent 3 Shot-bole injury common on several leaves 4 Several leaveswith shot-hole and elongated lesions 5 Several leaves with elongatedlesions 6 Several leaves with elongated lesions-2.5 cm. Long 7 Longlesions common on ~50% of leaves 8 Long lesions common on ~70% of leaves9 Most or all leaves with long lesions

The SWCB infested F₁ plants were also assessed for the length of stalkboring caused by SWCB. To determine the length of stalk boring, cornstalks of the corn plants were broken at approximately eye level and thetop portion was used to inspect for boring damage. The stalks were splitusing a double handled knife and the length of the tunnel bored out bySWCB was measured in centimeters (cm). In these experiments, the tunnellength was capped at ten centimeters (10 cm).

In addition, five (5) F₁ plants for each event were also infested withCEW to measure the amount of damage caused by CEW to the corn ear.Approximately forty (40) CEW nymphs were used to infest each plant andwere placed on the green silks of R₁ stage plants. Twenty-one (21) daysafter infestation, the developing ears were examined, and the damage wasrecorded as cm² ear damage.

Table 8 shows the mean leaf damage ratings for the F₁ events infestedwith FAW and SWCB, the stalk boring lengths caused by SWCB, and the eardamage caused by CEW, wherein “NT” indicates not tested.

TABLE 8 Mean leaf damage ratings of F₁ transgenic corn plants infestedwith FAW and SWCB, stalk boring lengths caused by SWCB, and ear damagecaused by CEW. CEW SWCB ear tunnel damage FAW SWCB length (cm² Event LDRLDR (cm) damage) MON 95379 0.30 1.00 0.00 3.25 Event 2 0.50 1.00 0.003.25 Event 3 0.30 1.00 0.00 0.00 Event 4 0.30 1.30 0.00 2.30 Event 51.00 1.00 0.00 1.00 Event 6 0.30 1.00 0.00 0.00 Event 7 0.80 0.50 0.003.00 Event 8 0.50 1.00 0.00 0.00 Event 9 1.30 0.80 0.00 0.25 Event 10 NTNT NT NT Event 11 NT NT NT NT Event 12 0.30 1.00 0.00 3.50 Event 13 NTNT NT NT Event 14 NT NT NT NT Event 15 NT NT NT NT Event 16 NT NT NT NTEvent 17 0.50 NT NT NT Event 18 0.00 1.30 0.00 2.50 Event 19 0.80 1.300.00 0.00 Event 20 NT NT NT NT Event 21 NT NT NT NT Event 22 NT NT NT NTEvent 23 NT NT NT NT Event 24 0.50 0.50 2.50 2.75 Event 25 0.00 1.502.25 0.00 Event 26 0.30 1.30 0.00 3.00 Event 27 0.50 1.50 2.50 1.25Event 28 0.30 1.00 1.00 1.75 Event 29 0.30 1.00 1.00 0.25 Event 30 NT NTNT NT Event 31 0.30 1.00 0.00 2.50 Event 32 0.50 1.00 0.00 0.25 Event 330.50 1.00 0.00 2.25 Event 34 0.30 1.00 0.00 1.50 Event 35 0.30 1.30 1.250.00 Event 36 NT NT NT NT Event 37 0.50 1.00 0.00 1.50 Event 38 1.301.00 0.00 0.50 Event 39 0.00 1.00 1.75 2.00 Event 40 0.30 0.50 1.25 1.75Event 41 0.00 0.80 0.00 2.25 Negative Control 7.80 9.00 10.00 13.25

As can be seen in Table 8, leaf damage to corn event MON 95379 wasminimal for both FAW and SWCB when compared to the negative controls.Essentially, once the insects started to feed on the event MON 95379 F₁leaf, expression of the Cry1B.868 and Cry1Da_7 insecticidal proteins inthe corn leaves containing event MON 95379 caused the insect to ceaseconsuming the leaf. SWCB tunneling was not observed in event MON 95379while the negative controls showed extensive tunneling. With respect toCEW car damage, the damage to the ear was much less compared to thenegative control, and was comparable to the ear damage observed inseveral commercially-available transgenic corn events. Infestation ofthe magnitude used in the F₁ assays was much higher than what is usuallyseen in nature. The F₁ assays demonstrated that corn event MON 95379provides superior control of FAW, SWCB, and CEW.

In the summer of 2016, the F_(j) progeny from the remaining thirty-nine(39) events after R₂/F₁ described in Example 2/Table 3 were assayed forresistance to FAW, CEW, and SWCB in field experiments using artificialinfestation. Multiple locations were used to assay resistance.

FAW resistance was assayed in three (3) locations: Jerseyville, IL;Thomasboro, IL; and Union City, TN. In each location, each event wasassayed in three (3) field plots using one (1) row per plot and thirty(30) seeds per row. Forty (40) FAW neonates were used to infest eachplant twice, at the early and mid-whorl stage (V4 and V7 vegetativestage). Leaf feeding damage ratings were assessed using the scale asprovided in Table 6.

SWCB resistance was assayed in three (3) locations: one (1) inJonesboro, AR and two (2) in Union City, TN. In each location, eachevent was assayed in three (3) field plots using one (1) row per plotand thirty (30) seeds per row. Thirty (30) SWCB neonates were used toinfest each plant at the mid-whorl stage (V7-V8). At the time of fiftypercent (50%) pollen shed, the plants were infested again with thirty(30) SWCB neonates per plant. Stalk tunneling damage was assessed aspreviously described.

CEW resistance was assayed in five (5) locations: Jerseyville, IL,Jonesboro, AR, Monmouth, IL, Thomasboro, IL, and Union City, TN. In eachlocation, each event was assayed in three (3) field plots using one (1)row per plot and thirty (30) seeds per row. Plants were infested whenthe silks were fresh and green, and some ear formation had started (R1through R3 stage). CEW egg strips were used for infestation. Each stripcontained approximately forty (40) eggs. One (1) strip was placedbetween the ear and stalk of each plant, with the eggs facing ear andclose to the silks. Evaluation of ear damage was determined twenty-one(21) to twenty-eight (28) days after infestations. By this time, theinsect has progressed from larval to pupal stage. Damage to the ears wasmeasured as previously described.

For FAW and SWCB, data from all three (3) locations was used. For CEW,due to various field conditions, only data from Jonesboro, AR could beused. Table 9 shows the mean FAW leaf damage ratings, the SWCB tunnellengths, and the CEW ear damage measurements for each of the testedevents and the negative control.

TABLE 9 Mean FAW leaf damage ratings, SWCB tunnel length, and CEW eardamage for 2016 field efficacy trials. SWCB CEW Tunnel Ear FAW LengthDamage Event LDR (cm) (cm²) MON 95379 1.56 0.37 2.86 Event 2 1.28 1.844.41 Event 3 1.33 0.14 3.28 Event 4 2.01 0.11 3.00 Event 5 1.83 0.894.09 Event 6 1.46 1.09 2.89 Event 7 1.67 0.39 3.40 Event 8 1.44 0.723.19 Event 9 1.59 0.00 3.98 Event 10 2.85 0.35 2.96 Event 11 1.50 2.592.84 Event 12 1.39 0.42 3.04 Event 13 1.74 0.00 3.34 Event 14 2.52 1.223.01 Event 15 1.53 0.22 3.09 Event 16 1.50 1.61 3.24 Event 17 2.31 1.722.80 Event 18 1.70 0.13 4.02 Event 19 1.28 1.00 3.96 Event 20 1.28 0.522.79 Event 21 1.39 0.39 3.55 Event 22 1.72 0.94 4.34 Event 23 1.86 0.223.04 Event 24 1.93 0.00 3.60 Event 25 1.57 0.06 2.28 Event 26 1.65 0.002.28 Event 27 1.65 0.28 2.73 Event 28 1.63 2.72 3.60 Event 29 2.62 0.783.42 Event 30 4.78 12.59 5.25 Event 31 1.78 1.81 3.79 Event 32 1.63 0.003.08 Event 33 1.37 0.00 3.40 Event 34 1.80 0.19 3.20 Event 35 2.20 0.843.99 Event 36 1.96 0.22 3.78 Event 37 1.35 0.06 3.07 Event 38 1.22 0.273.94 Event 39 1.46 0.14 2.87 Negative Control 7.15 7.21 32.25

As demonstrated in Table 9, corn event MON 95379 provided excellentcontrol of FAW, SWCB, and CEW when compared to the negative control. Thelevel of infestation in these assays was much higher than what wouldnormally be encountered in the field under natural conditions,demonstrating the superior performance of event MON 95379 under highinsect pressure.

During concurrent field trials and Cre-excision of the selectioncassette and the production of Gold Standard Seed, furthercharacterization of the events was performed. As a result of extensivemolecular characterization, efficacy, expression, and agronomic studies,events were dropped from testing, leaving two (2) events: Event 1 andMON 95379. Event 1 was de-prioritized based on observed yield drag inagronomic studies, leaving event MON 95379 for advancement.

During the 2016 to 2017 growing season in Argentina, event MON 95379 wasassayed for resistance to FAW, CEW, and SCB in temperate and subtropicalregions under natural infestation conditions. FAW leaf damage ratingswere determined for event MON 95379 grown in the sub-tropical region ofArgentina using the scale provided in Table 6. SCB tunneling data wasobtained for event MON 95379 from two (2) locations in the temperateregion of Argentina. CEW ear damage data was obtained for event MON95379 from two (2) locations in the temperate region and three (3)locations in the subtropical regions of Argentina. Table 11 shows themean FAW leaf damage ratings, SCB tunnel length, and CEW ear damageunder natural infestation conditions for event MON 95379 and a negativecontrol during the 2016-2017 Argentina growing seasons.

TABLE 10 Mean FAW leaf damage ratings, SCB tunnel length, and CEW eardamage for 2016-2017 Argentina field efficacy trials. CEW Ear FAW SCBTunnel Damage Event (LDR) Length (cm) (cm²) MON 95379 1.27 0.00 1.32Negative Control 7.48 4.43 5.83

As can be seen in Table 10, event MON 95379 provided resistance to FAW.SCB, and CEW when compared to the negative control under naturalinfestation conditions in Argentina.

Event MON 95379 was also evaluated for resistance against FAW resistantto a commercially-available corn event (MON89034, which expressesCry1a.105 and Cry2Ab2) over three (3) growing seasons in Puerto Rico(January 2016, July 2016, and January 2017). Table 11 shows the meanleaf damage ratings based upon the scale presented in Table 6 for eachof the three (3) growing seasons compared with event MON89034 and thenegative control.

TABLE 11 Mean leaf damage ratings for event MON 95379 and event MON89034naturally-infested with event MON89034-resistant FAW. Jan. Jul. Jan.Event 2016 2016 2017 MON 95379 2.30 2.22 1.40 MON89034 5.68 4.54 7.36Negative Control 8.73 6.84 9.00

As can be seen in Table 11, corn event MON 95379 demonstrated resistanceto event MON89034-resistant FAW under high natural pressure relative tothe negative control.

In the summer of 2017, event MON 95379 was evaluated for resistanceagainst FAW, SWCB, and CEW in the United States using methods similar tothat described for the summer of 2016. FAW resistance was assayed atthree (3) locations: Jerseyville, IL; Thomasboro, IL; and Monmouth, IL.In each location, each event was assayed in three (3) field plots usingone (1) row per plot and thirty (30) seeds per row. Forty (40) FAWneonates were used to infest each plant two times. The first infestationoccurred around V5 stage. The second infestation for plants in Monmouth.IL and Jerseyville. IL occurred around V8 stage. Due to a low hatch rateand poor weather, a second infestation was not possible in Thomasboro,IL. FAW leaf feeding damage ratings were assessed using the scale asprovided in Table 6.

SWCB resistance was assayed at three (3) locations, one (1) inJonesboro, AR and two (2) in Union City, IL. In each location, eachevent was assayed in three (3) field plots using one (1) row per plotand thirty (30) seeds per row. Thirty (30) SWCB neonates were used toinfest each plant two times. Under normal conditions, the first infestis performed at the mid-whorl stage (V7-V8) in half of the row, butinfestation was delayed about a week. Regardless, strong insect pressurewas established. At the time of fifty percent (50%) pollen shed thesecond half of the row of plants were infested with thirty (30) SWCBneonates per plant. Stalk tunneling damage was assessed as previouslydescribed.

CEW resistance was assayed at six (6) locations: Jerseyville, IL,Jonesboro, AR, Paragould, AR, Monmouth, IL, and two locations in UnionCity, TN. In each location, each event was assayed in three (3) fieldplots using one (1) row per plot and thirty (30) seeds per row. Due to ashortage of insects, infestations in Monmouth, IL and Jerseyville, ILwere infested two (2) to three (3) weeks later than when silks are freshand green. In Monmouth, approximately twenty-two (22) neonates were usedto infest each plant. In Jerseyville, IL, twenty-three (23) totwenty-four (24) neonates were used to infest partially opened cornears. In Jonesboro, AR, one (1) of the three (3) rows receivedapproximately thirty (30) neonates per plant, and the other two (2) rowsreceived sixteen (16) to (18) neonates per plant. In Paragould, AR, allthree (3) rows received approximately thirty (30) neonates per plant.Infestation was delayed in the two locations of Union City, TN due toinsect availability. Both locations received eighteen (18) neonates perplant. Evaluation of ear damage was determined twenty-one (21) totwenty-eight (28) days after infestations. Damage to the ears wasexpressed as previously described. Artificial infestations wereconducted on both marker and marker-free event MON 95379 plants. Inaddition, assays were also conducted using the natural insect pressureat the locations for the marker-containing event MON 95379 plants.Tables 12 and 13 show the FAW leaf damage ratings. SWCB tunnel lengths,and the CEW ear damage for marker-containing and marker-free event MON95379 plants.

TABLE 12 Mean FAW leaf damage ratings, SWCB tunnel length, and CEW eardamage for event MON 95379 plants before Cre-excision of the selectionmarker under conditions of artificial and natural infestation. BeforeCre-excision of CP4 Marker SWCB Artificial FAW FAW CEW CEW InfestationArtificial Natural Artificial Natural Tunnel Infestation InfestationInfestation Infestation Length Event (LDR) (LDR) (cm2) (cm2) (cm) MON95379 1.17 1.15 4.93 5.81 0.00 Negative 7.08 8.15 8.49 14.57 14.34Control

TABLE 13 Mean FAW leaf damage ratings, SWCB tunnel length, and CEW eardamage for marker-free event MON 95379 plants under artificialinfestation. After Cre-excision of CP4 Marker SWCB Artificial FAW CEWInfestation Artificial Artificial Tunnel Infestation Infestation LengthEvent (LDR) (cm2) (cm) MON 95379 1.20 5.39 0.27 Negative Control 7.088.49 14.34

As can be seen in Tables 12 and 13, event MON 95379 provided resistanceagainst FAW, SWCB, and CEW under artificial (marker and marker-free) andnatural (marker-free) infestation conditions.

In 2018, a hybrid cross of event MON 95379 with event MON89034 wasassayed for resistance to FAW in a Brazil field trial under naturalinfestation conditions. The field trial was conducted in Santa Helena deGoiás, State of Goiás. In this location there are FAW populationsresistant to the transgenic corn event MON89034. Transgenic corn plantscorresponding to the cross of events MON 95379×MON89034, event MON89034,and a conventional corn plant (negative control) were planted. At V6stage, leaf damage rating scores were determined for sixty (60) plantscorresponding to the cross of events MON 95379×MON89034, thirty (30)plants corresponding to event MON89034, and thirty (30) negativecontrols using the scale presented in Table 6. In addition, the numberof FAW neonates, larvae greater than two millimeters (2 mm) and lessthan or equal to 1.5 centimeters, and larvae greater than 1.5centimeters were recorded for each plant. Table 14 shows the mean leafdamage ratings for the cross of events MON 95379×MON89034, eventMON89034, and the negative control, along with the numbers of neonatesand larvae observed on the corn plants.

TABLE 14 Mean FAW leaf damage rating and number of neonates and larvaefrom Brazil, 2018 field trials for the cross of events MON 95379 ×MON89034, event MON89034 and negative control. FAW Larvae >2 mm LarvaeEvent (LDR) Neonates and ≤1.5 cm >1.5 cm MON 95379 × 0.62 0 0 MON89034MON89034 1.47 6 16 0 Negative Control 5.20 0 22 9

As can be seen in Table 14, the cross of events MON 95379×MON89034provided resistance to FAW under natural infestation conditions relativeto the negative control. The cross of events MON 95379×MON89034 alsoperformed better than event MON89034 under conditions where eventMON89034-resistant FAW are within the population of FAW. With respect toneonates and larvae, none were observed on the plants corresponding tothe cross of events MON 95379×MON89034. Neonates and larvae between two(2) millimeters and one and a half (1.5) centimeters were observed onevent MON89034 plants. The negative control plants were observed to haveeven more larvae than event MON89034 plants, and had larvae that hadgrown greater than 1.5 centimeters.

Example 5 Assay of Activity of Corn Event MON 95379 Against LesserCornstalk Borer

This Example describes the assay of activity of transgenic corn eventMON 95379 against the Lepidopteran insect pest, Lesser Cornstalk Borer(LSCB. Elasmopalpus lignosellus).

Event MON 95379 was grown in a greenhouse along with negative controlplants and infested with LSCB neonates. Ten (10) event MON 95379 plantsand nine (9) negative control plants were grown in individual pots. Nine(9) days after planting, each plant was infested with ten (10) LSCBneonates per plant. Twenty-two (22) days after infestation, the plantswere examined and rated for damage using a 0-4 damage rating scale aspresented in Table 15.

TABLE 15 LSCB plant damage rating scale. LSCB Damage Severity of RatingInjury Description 0 No damage Plants without injury 1 Slight injuryPlants with scratches in leaves and/or stalk 2 Average damage Plantswith stalk parts damaged 3 Serious damage Plants with stalk parts damageand dead hear symptoms 4 Dead plant Dead plants

The resulting LSCB damage ratings for each plant is presented in Table16.

TABLE 16 LSCB plant damage for each infested plant. Plant MON 95379Negative Control 1 0 4 2 0 2 3 0 3 4 0 3 5 0 4 6 0 3 7 0 3 8 0 4 9 0 410 0

As can be seen in Table 16, LSCB produced extensive damage to thenegative control plants, four (4) of which were rated as “Dead,” four(4) of which were rated as “Serious damage,” and only one (1) rated as“Average damage.” In contrast, the event MON 95379 LSCB infested plantsshowed no damage.

Transgenic corn event MON 95379 provides resistance to Lesser CornstalkBorer (LSCB, Elasmopalpus lignosellus).

Example 6 Corn Event MON 95379 Provides Consistent Yield and SimilarAgronomics to Untransformed LH244 Corn Plants

This Example demonstrates that transgenic corn event MON 95379 providesconsistent yields and similar agronomics in the field to untransformedLH244 corn plants.

Field trials were conducted with plants corresponding to event MON 95379prior to Cre-excision of the glyphosate selection cassette to determinevarious aspects of yield and agronomics in comparison to control plants.Measurements of yield were calculated and expressed as bushels per acre(bu/acre). Plant height and ear height were measured in inches (in).Fifty percent (50%) pollen shed and fifty percent (50%) silking wereexpressed as days after planting (DAP). Test weight, which is ameasurement of bulk density, or the weight of a unit volume, of grainwas expressed in pounds per bushel (lb/bu). The USDA established thestandard test weight of a bushel of corn as fifty-six pounds per bushel(56 lb/bu) based upon a 15.5% moisture content. The percent moisture ofthe corn kernel was expressed on a wet weight basis. The moisturecontent is the amount of water in the seed and is usually expressed as apercentage. It can be expressed on either a wet weight basis (where itis expressed as a percentage of the fresh weight of the seed) or on adry weight basis (where it is expressed as a percentage of the dryweight of the seed). Determination of percent moisture is destructive tothe seed. Percent moisture (wet basis) can be calculated with the simpleformula:M _(wb)=(W _(w) /W _(w) +W _(d))×100Where W_(w) is equal to the weight of the water and W_(d) is equal tothe weight of the dry matter.

In the growing season of 2016 in the United States, yield and agronomicmeasures were determined for event MON 95379 inbreds and hybridspre-Cre-excision of the glyphosate maker cassette. Tables 17 and 18 showthe yield and agronomic characteristics measured for event MON 95379inbreds and hybrids, respectively. The negative control plants for theinbred comparisons was untransformed variety LH244. Hybrids containingevent MON 95379 were created by cross pollinating the inbred event MON95379 with corn variety 93IDI3, and the untransformed control was anLH244×93IDI3 cross.

TABLE 17 Yield and agronomics for event MON 95379 inbreds relative tonon-transgenic controls. Plant 50% Pollen Yield Height Ear Height Shed50% Silking (bu/acre) (in) (in) (DAP) (DAP) Event Mean SE Mean SE MeanSE Mean SE Mean SE MON 111.08 5.17 85.40 2.72 41.29 1.45 65.11 2.0065.77 2.05 95379 LH244 114.18 5.02 82.05 2.32 38.73 1.33 62.60 1.7963.54 1.82

TABLE 18 Yield and agronomic for event MON 95379 hybrids relative tonon-transgenic controls. Yield Test Weight Percent (bu/acre) (lb/bu)Moisture Event Mean SE Mean SE Mean SE MON 95379 x 93IDI3 203.48 7.0458.78 0.61 16.04 0.42 LH244 x 93IDI3 196.45 6.81 58.31 0.61 15.89 0.43

As can be seen in Tables 17 and 18, the yield and other agronomicmeasures for event MON 95379 in the 2016 United States field trials wererelatively the same for both inbreds and hybrids relative to thecontrols. The variability between the inbreds and hybrids and theirrespective controls was within acceptable limits and demonstrate therewere no negative impacts on yield and other agronomic characteristicscaused by insertion of the T-DNA into the corn genome of event MON95379.

Yield and agronomics were also studied in Argentina during the 2016 to2017 growing season for event MON 95379 inbreds and hybridspre-Cre-excision of the glyphosate marker cassette. Tables 19 and 20show the yield and agronomic characteristics measured for event MON95379 inbreds. The negative control plants for the inbred comparisonswas untransformed variety LH244. Hybrids containing event MON 95379 werecreated by cross-pollinating events MON89034×MON895379. The transgeniccontrol was event MON88017×event MON89034. The non-transgenic controlwas a LH244×93IDI3 cross. Table 21 shows the yield and agronomiccharacteristics measured for event MON 95379 hybrids, wherein “NC”indicates not calculated.

TABLE 19 Yield and agronomics for event MON 95379 inbreds relative tonon-transgenic controls. Plant Ear 50% Pollen Yield Height Height Shed50% Silking (bu/acre) (in) (in) (DAP) (DAP) Event Mean SE Mean SE MeanSE Mean SE Mean SE MON 113.39 4.57 73.89 1.05 38.38 0.95 64.32 0.6064.68 0.62 95379 LH244 105.49 NC 71.69 NC 36.66 NC 64.86 NC 65.17 NC

TABLE 20 Yield and agronomics for event MON 95379 inbreds relative tonon-transgenic controls. Test Weight (lb/bu) Percent Moisture Event MeanSE Mean SE MON 95379 59.28 0.10 16.54 0.26 LH244 59.32 NC 16.01 NC

TABLE 21 Yield and agronomics for event MON 95379 hybrids relative totransgenic and non-transgenic controls. Yield Test Weight Percent(bu/acre) (lb/bu) Moisture Event Mean SE Mean SE Mean SE MON89034 x MON95379 165.80 5.94 60.06 0.20 15.12 0.18 MON88017 x MON89034 164.87 5.9459.62 0.20 15.45 0.18 LH244 x 93IDI3 166.82 NC 59.70 NC 15.38 NC

As can be seen in Tables 19 through 21, the measures of yield and otheragronomic characteristics were relatively the same for event MON 95379inbreds and hybrids relative to the controls.

In 2017, yield and agronomics were again measured in field trials in theUnited States for event MON 95379 inbreds and hybrids pre-Cre-excisionof the glyphosate marker cassette. Inbred and hybrid controls weresimilar to those used in the 2016 United States field trials. Table 22shows the yield and agronomic characteristics for event MON 95379inbreds relative to non-transgenic controls, and Tables 23 and 24 showthe yield and agronomic characteristics measured for event MON 95379hybrids relative to non-transgenic controls in the 2017 United Statesfield trials.

TABLE 22 Yield and agronomics for event MON 95379 inbreds relative tonon-transgenic controls. Yield Test Weight Percent (bu/acre) (lb/bu)Moisture Event Mean SE Mean SE Mean SE MON 95379 116.49 5.45 57.86 0.5921.16 0.76 LH244 124.46 5.39 58.60 0.58 20.57 0.76

TABLE 23 Yield and agronomics for event MON 95379 inbreds relative tonon-transgenic controls. 50% Plant Ear Pollen 50% Yield Height HeightShed Silking (bu/acre) (in) (in) (DAP) (DAP) Event Mean SE Mean SE MeanSE Mean SE Mean SE MON 213.15 4.84 99.14 2.50 45.27 1.77 56.65 0.9357.17 1.06 95379 x 93IDI3 LH244 x 93IDI3 217.86 4.44 97.42 2.28 45.611.45 55.87 0.91 56.47 1.04

TABLE 24 Yield and agronomic for event MON 95379 hybrids relative tonon-transgenic controls. Test Weight (lb/bu) Percent Moisture Event MeanSE Mean SE MON 95379 × 93IDI3 57.98 0.37 19.73 0.51 LH244 × 93IDI3 57.530.36 19.74 0.50

As can be seen in Tables 22 through 24, the yield and other agronomicproperties event MON 95379 demonstrated in the 2017 United Stated fieldtrials were similar to the untransformed controls for both inbred andhybrid lines.

During the 2018 to 2019 growing season in Argentina, agronomics andyield were measured in field trials for event MON 95379 inbreds andhybrids post-Cre-excision of the glyphosate marker cassette. Inbredcontrols were similar to those used in the 2017 United States fieldtrials. The hybrids were produced through crosses with the elite variety80IDM2. Tables 25 and 26 show the yield and agronomic characteristicsfor event MON 95379 inbreds relative to non-transgenic controls, andTable 27 shows the yield and agronomic characteristics measured forevent MON 95379 hybrids relative to non-transgenic controls in the 2018to 2019 Argentina field trials.

TABLE 25 Yield and agronomics for event MON 95379 inbreds relative tonon-transgenic controls. 50% Plant Ear Pollen 50% Yield Height HeightShed Silking (bu/acre) (in) (in) (DAP) (DAP) Event Mean SE Mean SE MeanSE Mean SE Mean SE MON 95379  92.81 7.30 85.14 2.45 40.57 2.06 62.740.61 63.77 0.65 LH244 103.46 7.68 83.93 1.72 38.57 1.24 62.93 0.61 63.880.71

TABLE 26 Yield and agronomics for event MON 95379 inbreds relative tonon-transgenic controls. Test Weight Percent (Ib/bu) Moisture Event MeanSE Mean SE MON 59.59 1.52 17.49 0.96 95379 LH244 60.34 0.97 17.24 0.69

TABLE 27 Yield and agronomics for event MON 95379 hybrids relative tonon-transgenic controls. Yield Test Weight Percent (bu/acre) (lb/bu)Moisture Event Mean SE Mean SE Mean SE MON 95379 x 206.86 8.37 59.420.46 19.03 0.84 80IDM2 LH244 x 207.81 8.11 59.22 0.45 19.12 0.83 80IDM2

As can be seen in Tables 25 through 27, the yield and other agronomicproperties event MON 95379 demonstrated in the 2017 to 2018 Argentinafield trials were similar to the untransformed controls for both inbredand hybrid lines.

Thus, in sum, corn event MON 95379 demonstrated similar yield and otheragronomic properties over four (4) separate growing seasons in theUnited States and Argentina. Event MON 95379 does not negatively affectyield or cause a change in other agronomic properties measured comparedto non-transgenic and transgenic controls.

Example 7 Corn Event MON 95379 Event-Specific Endpoint TAQMAN® Assays

The following Example describes methods useful in identifying thepresence of event MON 95379 in a corn sample. A pair of PCR primers anda probe were designed for the purpose of identifying the unique junctionformed between the corn genomic DNA and the inserted DNA of event MON95379 in an event-specific endpoint TAQMAN® PCR. Examples of conditionsutilized for identifying the presence of event MON 95379 in a cornsample in an event-specific endpoint TAQMAN® PCR are described in Table28 and Table 29.

The sequence of the oligonucleotide forward primer SQ21529 (SEQ IDNO:15) is identical to the nucleotide sequence corresponding topositions 833-852 of SEQ ID NO:10. The sequence of the oligonucleotidereverse primer SQ21524 (SEQ ID NO:16) is identical to the reversecomplement of the nucleotide sequence corresponding to positions 905-934of SEQ ID NO:10. The sequence of the oligonucleotide probe PB10269 (SEQID NO:17) is identical to the reverse complement of the nucleotidesequence corresponding to positions 886-901 of SEQ ID NO:10. The primersSQ21529 (SEQ ID NO:15) and SQ21524 (SEQ ID NO:16) with probe PB10269(SEQ ID NO:17), which may be fluorescently labeled (e.g., a 6-FAM™fluorescent label), can be used in an endpoint TAQMAN® PCR assay toidentify the presence of DNA derived from event MON 95379 in a sample.

In addition to SQ21529 (SEQ ID NO:15), SQ21524 (SEQ ID NO:16), andPB10269 (SEQ ID NO:17), it should be apparent to persons skilled in theart that other primers and/or probes can be designed to either amplifyor hybridize to sequences within SEQ ID NO:10 which are unique to, anduseful for, detecting the presence of DNA derived from event MON 95379in a sample.

Following standard molecular biology laboratory practices, PCR assaysfor event identification were developed for detection of event MON 95379in a sample. Parameters of either a standard PCR assay or a TAQMAN® PCRassay were optimized with each set of primer pairs and probes (e.g.,probes labeled with a fluorescent tag such as 6-FAM™) used to detect thepresence of DNA derived from event MON 95379 in a sample. A control forthe PCR reaction includes internal control primers and an internalcontrol probe (e.g., VIC®-labeled) specific to a region within the corngenome that is used as an internal control, and are primers SQ20222 (SEQID NO:18), SQ20221 (SEQ ID NO:19), and VIC® labeled probe PB50237 (SEQID NO:20).

Generally, the parameters which were optimized for detection of eventMON 95379 in a sample included primer and probe concentration, amount oftemplated DNA, and PCR amplification cycling parameters. The controlsfor this analysis include a positive control from corn containing eventMON 95379, a negative control from non-transgenic corn, and a negativecontrol that contains no template DNA.

TABLE 28 MON 95379 event-specific endpoint TAQMAN ® PCR reactioncomponents. Stock Final Concentration Volume Concentration Step Reagent(μM) (μl) (μM) Comments Reaction volume 5 1 2 × Master Mix 2.28 1 ×final concentration 2 Event Specific Primer 100 0.05 0.9 SQ51219 3 EventSpecific Primer 100 0.05 0.9 SQ21524 4 Event Specific 6FAM ™ 100 0.010.2 Probe is light probe PB10269 sensitive 5 Internal Control Primer 1000.05 0.9 SQ20222 6 Internal Control Primer 100 0.05 0.9 SQ20221 7Internal Control VIC ® 100 0.01 0.2 Probe is light probe PB50237sensitive 8 Extracted DNA (template): 2.5 Separate reactions LeafSamples to be are made for each analyzed template. Negative control(non- transgenic DNA) Negative water control (No template control)Positive Qualitative control(s) MON 95379 DNA

TABLE 29 Endpoint TAQMAN ® thermocycler conditions. Step Number No. ofCycles Settings 1 1 95° C. 20 seconds 2 40 95° C. 3 seconds 60° C. 20seconds 3 1 10° C.

Example 8 Assays for Determining Zygosity for Event MON 95379 UsingTAQMAN® and Detection of the Insect Toxin Transgenes

The following Example describes methods useful in identifying thezygosity of event MON 95379 and detection of the insect toxin transgenesin event MON 95379 in a corn sample. Pairs of PCR primers and a probeare designed for the purpose of identifying specific properties ofalleles positive and negative for the T-DNA insertion that gave rise toevent MON 95379.

A zygosity assay is useful for determining if a plant comprising anevent is homozygous for the event DNA (i.e., comprising the exogenousDNA in the same location on each chromosome of the chromosomal pair),heterozygous for the event DNA (i.e., comprising the exogenous DNA ononly one chromosome of the chromosomal pair), or wild-type (i.e., nullfor the event DNA).

An endpoint TAQMAN® thermal amplification method was used to develop azygosity assay for event MON 95379. The assay uses a primer pair and aprobe to detect amplicons corresponding to one of the two insect toxincoding sequences encoding Cry1B.868 and Cry1Da_7 comprised within theT-DNA used to generate corn event MON 95379. In addition, a primer pairand probe are used to detect a single-copy internal control that islocated within the corn genome and is known to be present as ahomozygous allele.

For this assay two (2) primer pairs and two (2) probes were mixedtogether with the sample. The DNA primers used in the zygosity assaywhich detects the presence of the Cry1B.868 toxin coding sequence wereprimers SQ50998 (SEQ ID NO:21) and SQ50997 (SEQ ID NO:22). TheVIC®-labeled DNA probe used in the zygosity assay which detects thepresence of the Cry1B.868 toxin coding sequence was PB54340 (SEQ IDNO:23). The DNA primers used in the zygosity assay which detect thepresence of the Cry1Da_7 toxin coding sequence were primers SQ50485 (SEQID NO:24) and SQ50484 (SEQ ID NO:25). The VIC®-labeled DNA probe used inthe zygosity assay which detects the presence of the Cry1Da_7 toxincoding sequence was PB50138 (SEQ ID NO:26). Both zygosity detectionassays use the same internal control. The primers for the internalcontrol were SQ20222 (SEQ ID NO: 18) and SQ20221 (SEQ ID NO: 19), andthe 6FAM™-labeled probe for the internal control was PB50237 (SEQ IDNO:20). The DNA primers and probe for either Cry1B.868 or Cry1 Da_7 weremixed with the primers and probe for the internal control as shown inTables 30 and 31.

TABLE 30 Corn event MON 95379 zygosity TAQMAN ® PCR for the detection ofCry1B.868. Stock Final Concentration Volume Concentration Step Reagent(μl) (μl) (μM) Comments Reaction volume 5 1 2 × Master Mix 2.4 1 × finalconcentration 2 Cry1B.868 specific primer 100 0.0225 0.45 SQ50998 3Cry1B.868 specific primer 100 0.0225 0.45 SQ50997 4 Cry1B.868 6FAM ™probe 100 0.005 0.1 Probe is light sensitive PB50340 5 Internal ControlPrimer 100 0.0225 0.45 SQ20222 6 Internal Control Primer 100 0.0225 0.45SQ20221 7 Internal Control VIC ® probe 100 0.005 0.1 Probe is lightsensitive PB50237 8 Extracted DNA (template): 2.5 Separate reactions areLeaf Samples to be analyzed made for each template. Negative control(non- transgenic DNA) Negative water control (No template control)Positive copy number control(s) Cry1B.868

TABLE 31 Corn event MON 95379 zygosity TAQMAN ® PCR for the detection ofCry1Da_7. Stock Final Concentration Volume Concentration Step Reagent(μl) (μl) (μM) Comments Reaction volume 5 1 2 × Master Mix 2.4 1 × finalconcentration 2 Cry1Da_7 specific Primer 100 0.0225 0.45 SQ50485 3Cry1Da_7 specific Primer 100 0.0225 0.45 SQ50484 4 Cry1Da_7 specific6FAM ™ 100 0.005 0.1 Probe is light sensitive probe PB50138 5 InternalControl Primer 100 0.0225 0.45 SQ20222 6 Internal Control Primer 1000.0225 0.45 SQ20221 7 Internal Control VIC ® probe 100 0.005 0.1 Probeis light sensitive PB50138 8 Extracted DNA (template): 2.5 Separatereactions are Leaf Samples to be analyzed made for each template.Negative control (non- transgenic DNA) Negative water control (Notemplate control) Positive copy number control(s) Cry1Da_7

Separate reactions are mixed using DNA derived from a leaf sample forwhich zygosity is not known, a negative control of DNA derived from anuntransformed corn plant, a negative control lacking DNA, and a positivecontrol using DNA derived from a transgenic plant homozygous forCry1B.868 or Cry1Da_7, depending upon which toxin coding sequence isused for detection. The reactions are then subjected to the thermalcycles presented in Table 32.

TABLE 32 Zygosity TAQMAN ® Thermocycler conditions. Step Number No. ofCycles Settings 1 1 95° C. 20 seconds 2 40 95° C. 3 seconds 60° C. 20seconds 3 1 10° C.

After amplification, the cycle thresholds (Ct values) were determinedfor the amplicon corresponding to the toxin coding sequence and thesingle-copy, homozygous internal standard. The difference (ΔCt) betweenthe Ct value of the single-copy, homozygous internal standard ampliconand the Ct value of the toxin coding sequence amplicon was determined.With respect to zygosity, a ΔCt of around zero (0) indicatedhomozygosity of the inserted event MON 95379 T-DNA and ΔCt of around one(1) indicated heterozygosity of the inserted event MON 95379 T-DNA. Lackof an amplicon corresponding to the insect toxin coding sequenceindicated the sample is null for the inserted event MON 95379 T-DNA. TheCt values in the TAQMAN® thermal amplification method will have somevariability due to multiple factors such as amplification efficiency andideal annealing temperatures. Therefore, the range of “about one (1)” isdefined as a ΔCt of 0.75 to 1.25.

For each progeny derived from a cross with event MON 95379, assays wereperformed for both toxin coding sequences to assure accuracy in thedetermination of zygosity of the progeny.

Example 9 Assays for Determining Zygosity for Corn Event MON 95379 UsingTAQMAN®

The following Example describes a method useful in identifying thezygosity of event MON 95379 in a corn sample.

Pairs of PCR primers and a probe are designed for the purpose ofidentifying specific properties of alleles positive and negative for theT-DNA insertion that gave rise to event MON 95379. Examples ofconditions that may be used in an event-specific zygosity TAQMAN® PCRare provide in Tables 33 and 34. For this assay, three primers and twoprobes were mixed together with the sample. The DNA primers used in thezygosity assay were primers SQ50219 (SEQ ID NO:15). SQ21524 (SEQ IDNO:16), and PWTDNA (SEQ ID NO:27). The probes used in the zygosity assaywere 6FAM™-labeled probe PB10269 (SEQ ID NO:17) and VIC®-labeled probePRWTDNA (SEQ ID NO:28). Primers SQ50219 (SEQ ID NO:15) and SQ21524 (SEQID NO:16) and the 6FAM™-labeled probe PB10269 (SEQ ID NO:17) arediagnostic for event MON 95379 DNA. SQ50219 (SEQ ID NO:15) and PWTDNA(SEQ ID NO:27) and the VIC®-labeled probe PRWTDNA (SEQ ID NO:28) arediagnostic when there is no copy of event MON 95379; i.e., they arediagnostic for the wild type allele.

When the three primers and two probes are mixed together in a PCRreaction with DNA extracted from a plant heterozygous for event MON95379, there is a fluorescent signal from both the 6FAM™-labeled probePB10269 (SEQ ID NO:17) and the VIC®-labeled probe PRWTDNA (SEQ ID NO:28)which is indicative of and diagnostic for a plant heterozygous for eventMON 95379. When the three primers and two probes are mixed together in aPCR reaction with DNA extracted from a plant homozygous for event MON95379, there is a fluorescent signal from only the 6FAM™-labeled probePB10269 (SEQ ID NO:17) and not the VIC®-labeled probe PRWTDNA (SEQ IDNO:28). When the three primers and the two probes are mixed together ina PCR reaction with DNA extracted from a plant which is null for eventMON 95379 (i.e., the wild-type), there is a fluorescent signal from onlythe VIC®-labeled probe PRWTDNA (SEQ ID NO:28). The template DNA samplesand controls for this analysis were a positive control from corncontaining event MON 95379 DNA (from both a known homozygous and a knownheterozygous sample), a negative control from non-transgenic corn and anegative control that contains no template DNA.

TABLE 33 Event MON 95379 zygosity TAQMAN ® PCR Stock Final ConcentrationVolume Concentration Step Reagent (μl) (μl) (μM) Comments Reactionvolume 5 1 18 megohm water 0.33 Adjust for final volume 2 2 × Master Mix2.5 1 × final concentration 3 Event Specific Primer 100 0.05 0.9 SQ512194 Event Specific Primer 100 0.05 0.9 SQ21524 5 Event Specific 6FAM ™ 1000.01 0.2 Probe is light sensitive probe PB10269 6 WT allele Primer 1000.05 0.9 PNEGDNA 7 WT allele VIC ® probe 100 0.01 0.2 Probe is lightsensitive PRBNEGDNA 8 Extracted DNA 2.5 Separate reactions are(template): made for each template. Leaf Samples to be analyzed Negativecontrol (non- transgenic DNA) Negative water control (No templatecontrol) Positive Qualitative control(s) MON 95379 DNA

TABLE 34 Zygosity TAQMAN ® thermocycler conditions Step Number No. ofCycles Settings 1 1 95° C. 20 seconds 2 40 95° C. 3 seconds 60° C. 20seconds 3 1 10° C.

Example 10 Identification of Corn Event MON 95379 in any MON 95379Breeding Event

The following Example describes how one may identify the MON 95379 eventwithin progeny of any breeding activity using corn event MON 95379.

DNA primer pairs are used to produce an amplicon diagnostic for cornevent MON 95379. An amplicon diagnostic for event MON 95379 comprises atleast one junction sequence. The junction sequences for event MON 95379are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, and SEQ ID NO:8 ([1], [2], [3], [4], [5], [6],[7], and [8], respectively in FIG. 1 ). SEQ ID NO:1 is a fifty (50)nucleotide sequence representing the 5′ junction region of corn genomicDNA and the integrated transgenic expression cassette. SEQ ID NO:1 ispositioned in SEQ ID NO:10 at nucleotide position 838-887. SEQ ID NO:2is a fifty (50) nucleotide sequence representing the 3′ junction regionof corn genomic DNA and the integrated transgenic expression cassette.SEQ ID NO:2 is positioned in SEQ ID NO:10 at nucleotide position14156-14205. SEQ ID NO:3 is a one hundred (100) nucleotide sequencerepresenting the 5′ junction region of corn genomic DNA and theintegrated transgenic expression cassette. SEQ ID NO:3 is positioned inSEQ ID NO:10 at nucleotide position 813-912. SEQ ID NO:4 is a onehundred (100) nucleotide sequence representing the 3′ junction region ofcorn genomic DNA and the integrated transgenic expression cassette. SEQID NO:4 is positioned in SEQ ID NO:10 at nucleotide position14,131-14,230. SEQ ID NO:5 is a two hundred (200) nucleotide sequencerepresenting the 5′ junction region of corn genomic DNA and theintegrated transgenic expression cassette. SEQ ID NO:5 is positioned inSEQ ID NO:10 at nucleotide position 763-962. SEQ ID NO:6 is a twohundred (200) nucleotide sequence representing the 3′ junction region ofcorn genomic DNA and the integrated transgenic expression cassette. SEQID NO:6 is positioned in SEQ ID NO:10 at nucleotide position14,081-14,280. SEQ ID NO:7 is a one thousand one hundred sixty (1.160)nucleotide sequence representing the 5′ junction region of corn genomicDNA and the integrated transgenic expression cassette. SEQ ID NO:7 ispositioned in SEQ ID NO:10 at nucleotide positions 1-1,160. SEQ ID NO:8is a one thousand one hundred seventy eight (1,178) nucleotide sequencerepresenting the 3′ junction region of the integrated transgenicexpression cassette and the corn genomic DNA. SEQ ID NO:8 is positionedin SEQ ID NO:10 at nucleotide positions 14,039-15,216.

Primer pairs that will produce an amplicon diagnostic for event MON95379 include primer pairs based upon the flanking sequences (SEQ IDNO:11 and SEQ ID NO:12) and the inserted T-DNA (SEQ ID NO:9). To acquirea diagnostic amplicon in which SEQ ID NO:1, or SEQ ID NO:3, or SEQ IDNO:5, or SEQ ID NO:7 is found, one would design a forward primermolecule based upon the 5′ flanking corn genomic DNA (SEQ ID NO:11; frombases 1 to 862 of SEQ ID NO:10) and a reverse primer molecule based uponthe inserted T-DNA (SEQ ID NO:9; from positions 863 through 14.180 ofSEQ ID NO:10) in which the primer molecules are of sufficient length ofcontiguous nucleotides to specifically hybridize to SEQ ID NO:11 and SEQID NO:9. To acquire a diagnostic amplicon in which SEQ ID NO:2, or SEQID NO:4, or SEQ ID NO:6, or SEQ ID NO:8 is found, one would design aforward primer molecule based upon the inserted T-DNA (SEQ ID NO:9; frompositions 863 through 14,180 of SEQ ID NO:10) and a reverse primermolecule based upon the 3′ flanking corn genomic DNA (SEQ ID NO:12; frompositions 14,181 through 15,216 of SEQ ID NO:10) in which the primermolecules are of sufficient length of contiguous nucleotides tospecifically hybridize to SEQ ID NO:9 and SEQ ID NO:12.

For practical purposes, one should design primers which produceamplicons of a limited size range, preferably between 200 to 1000 bases.Smaller sized amplicons in general are more reliably produced in PCRreactions, allow for shorter cycle times, and can be easily separatedand visualized on agarose gels or adapted for use in endpointTAQMAN®-like assays. In addition, amplicons produced using said primerpairs can be cloned into vectors, propagated, isolated and sequenced, orcan be sequenced directly with methods well established in the art. Anyprimer pair derived from the combinations of SEQ ID NO:11 and SEQ IDNO:9 or SEQ ID NO:12 and SEQ ID NO:9 that are useful in a DNAamplification method to produce an amplicon diagnostic for event MON95379 or progeny thereof is an aspect of the present invention. Anysingle isolated DNA polynucleotide primer molecule comprising at leasteleven (11) contiguous nucleotides of SEQ ID NO:11, SEQ ID NO:9 or SEQID NO:12 or their complements that is useful in a DNA amplificationmethod to produce an amplicon diagnostic for event MON 95379 or progenythereof is an aspect of the present invention.

An example of the amplification conditions for this analysis isillustrated in Tables 28 and 29. Any modification of these methods orthe use of DNA primers homologous or complementary to SEQ ID NO:11 orSEQ ID NO:12, or DNA sequences of the genetic elements contained in thetransgene insert (SEQ ID NO:9) of event MON 95379, that produce anamplicon diagnostic for event MON 95379 is within the art. A diagnosticamplicon comprises a DNA molecule homologous or complementary to atleast one transgene/genomic junction DNA or a substantial portionthereof.

An analysis for an event MON 95379 containing plant tissue sample shouldinclude a positive tissue control from a plant that contains event MON95379, a negative control from a corn plant that does not contain eventMON 95379 (e.g., LH244), and a negative control that contains no corngenomic DNA. A primer pair will amplify an endogenous corn DNA moleculeand will serve as an internal control for the DNA amplificationconditions. Additional primer sequences can be selected from SEQ ID NO:11, SEQ ID NO: 12, or SEQ ID NO: 9 by those skilled in the art of DNAamplification methods. Conditions selected for the production of anamplicon by the methods shown in Table 28 and Table 29 may differ, butresult in an amplicon diagnostic for event MON 95379 DNA. The use of DNAprimer sequences within or with modifications to the methods of Table 28and Table 29 are within the scope of the invention. An amplicon producedby at least one DNA primer sequence derived from SEQ ID NO:11, SEQ IDNO:12, or SEQ ID NO:9 that is diagnostic for event MON 95379 is anaspect of the invention.

DNA detection kits that contain at least one DNA primer of sufficientlength of contiguous nucleotides derived from SEQ ID NO:11, SEQ IDNO:12, or SEQ ID NO:9 that, when used in a DNA amplification method,produces a diagnostic amplicon for event MON 95379 or its progeny is anaspect of the invention. A corn plant or seed, wherein its genome willproduce an amplicon diagnostic for event MON 95379, when tested in a DNAamplification method is an aspect of the invention. The assay for theevent MON 95379 amplicon can be performed by using an Applied BiosystemsGeneAmp™ PCR System 9700, Stratagene Robocycler®, Eppendorf®Mastercycler® Gradient thermocycler or any other amplification systemthat can be used to produce an amplicon diagnostic of event MON 95379 asshown in Table 29.

All publications and published patent documents cited in thisspecification, and which are material to the invention, are incorporatedherein by reference to the same extent as if each individual publicationor patent application was specifically and individually indicated to beincorporated by reference.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

What is claimed is:
 1. A recombinant DNA molecule derived from cornevent MON 95379, a representative sample of seed comprising said eventhaving been deposited as ATCC Accession No. PTA-125027, wherein therecombinant DNA molecule comprises the nucleotide sequence of SEQ IDNO:1, SEQ ID NO:2, or a complete complement thereof.
 2. A DNA moleculecomprising a polynucleotide segment of sufficient length to function asa DNA probe that hybridizes specifically under stringent hybridizationconditions with the recombinant DNA molecule of claim 1 in a sample,wherein detecting hybridization of said DNA molecule under saidstringent hybridization conditions is diagnostic for the presence ofcorn event MON 95379 DNA in said sample, and wherein the DNA moleculecomprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 or acomplete complement thereof.
 3. The DNA molecule of claim 2, wherein theDNA molecule comprises the nucleotide sequence of SEQ ID NO:1 or acomplete complement thereof.
 4. An amplicon comprising a recombinant DNAmolecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,and a complete complement thereof.
 5. A method of detecting the presenceof a DNA segment diagnostic for corn event MON 95379 DNA in a sample,said method comprising: a) contacting said sample with the DNA moleculeof claim 2; b) subjecting said sample and said DNA molecule to stringenthybridization conditions; and c) detecting hybridization of said DNAmolecule to said DNA in said sample, wherein said detection isdiagnostic for the presence of said corn event MON 95379 DNA in saidsample, and wherein said corn event MON 95379 DNA comprises a codingsequence for Cry1Da_7.
 6. A method of detecting the presence of a DNAsegment diagnostic for corn event MON 95379 DNA in a sample, said methodcomprising: a) contacting said sample with a pair of DNA moleculescapable of producing a DNA amplicon diagnostic for event MON 95379,wherein the amplicon comprises the nucleotide sequence of SEQ ID NO:1 orSEQ ID NO:2 or a complete complement thereof; b) performing anamplification reaction sufficient to produce the DNA amplicon; and c)detecting the presence of said DNA amplicon in said reaction, whereinsaid corn event MON 95379 DNA comprises a coding sequence for Cry1Da_7.